Network Pharmacology Analysis of Liquid-Cultured Armillaria ostoyae Mycelial Metabolites and Their Molecular Mechanism of Action against Gastric Cancer.

Armillaria sp. are traditional edible medicinal mushrooms with various health functions; however, the relationship between their composition and efficacy has not yet been determined. Here, the ethanol extract of liquid-cultured Armillaria ostoyae mycelia (AOME), a pure wild Armillaria sp. strain, was analyzed using UHPLC-QTOF/MS, network pharmacology, and molecular docking techniques. The obtained extract affects various metabolic pathways, such as JAK/STAT and PI3K/AKT. The extract also contains important compounds such as 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl] benzamide, isoliquiritigenin, and 7-hydroxycoumarin. Moreover, the extract targets key proteins, including EGFR, SCR, and IL6, to suppress the progression of gastric cancer, thereby synergistically inhibiting cancer development. The molecular docking analyses indicated that the main compounds stably bind to the target proteins. The final cell culture experimental data showed that the ethanol extract inhibited MGC-803 gastric cancer cells. In summary, our research revealed the beneficial components of AOME for treating gastric cancer and its associated molecular pathways. However, further research is needed to confirm its effectiveness and safety in gastric cancer patients.

Glucans from Armillaria luteo-virens: Structural Characterization and In Vivo Immunomodulatory Investigation under Different Administration Routes.

Polysaccharides fromArmillaria luteo-virens (ALP) were investigated for structural characterization and immunomodulatory activities. Three fractions (ALP-1, ALP-2, and ALP-3) were obtained with the yield of 2.4, 3.7, and 3.0 wt %, respectively. ALP-1 was proposed as a ß-(1 → 3)(1 → 6)-glucan with a triple-helix conformation; ALP-2 and ALP-3 were both identified as α-(1 → 4)(1 → 6)-glucan differing in their Mw and branching degree with a spherical conformation. The in vitro digestibility experiment and in vivo experiments using cyclophosphamide (CY)-treated mice demonstrated that intraperitoneal injection of α-glucan (1 mg·kg-1·day-1) and intragastric gavage of ß-glucan (10 mg·kg-1·day-1) both effectively restored the decrease in body weight, immune organ indexes, immune cell activities, serum immune marker levels, colonic short-chain fatty acids (SCFA) levels, and Bacteroidetes/Firmicutes ratio in immunosuppression mice. This study provides novel insights into the immunomodulatory activity of α- and ß-glucans under different administration routes, thereby promoting their application in both food and pharmaceutical areas.

Decoding the RNA virome of the tree parasite Armillaria provides new insights into the viral community of soil-borne fungi.

The globally distributed basidiomycete genus Armillaria includes wood decomposers that can act as opportunistic parasites, causing deadly root rot on woody plants. To test whether RNA viruses are involved in this opportunistic behaviour, a large isolate collection of five Armillaria species collected over 40 years in Switzerland from trees, dead wood and soil was analysed. De novo assembly of RNA-Seq data revealed 21 viruses, 14 of which belong to putative new species. Two dsRNA viruses and an unclassified Tymovirales are formally described for the first time for Armillaria. One mitovirus occurred with a high prevalence of 71.1%, while all other viruses were much less prevalent (0.6%-16.9%). About half of all viruses were found only in one fungal species, others occurred in 2-6 fungal species. Co-infections of 2-7 viruses per isolate were not uncommon (34.9%), and most viruses persisted circulating within fungal populations for decades. Some viruses were related to viruses associated with other Armillaria species, supporting the hypothesis that virus transmission can occur between different fungal species. Although no specific correlation between viruses and the fungal trophic strategy was found, this study opens new insights into viral diversity hidden in the soil microbiome.

Armillariella tabescens polysaccharide treated rats with oral ulcers through modulation of oral microbiota and activation of the Nrf2/HO-1 pathway.

We identified Armillariella tabescens polysaccharide (PAT-W), a compound isolated from a Chinese medicinal mushroom, as a potential novel oral ulcer (OU) drug. In treating OU rats with PAT-W, especially in the high-dose group, oral mucous tissue TNF-α, IL-1ß, and IL-6 levels were markedly reduced, and pathological morphology and oxidative stress were effectively improved. Western blot analysis showed that the PAT-W channel ameliorated OU mucous tissue damage, which depends on the activation of the Nrf2/HO-1 antioxidant signaling pathway. Furthermore, high-throughput sequencing results showed that PAT-W regulated the maladjustment of the oral microbiota caused by OU. Therefore, based on the new viewpoint of activating the Nrf2/HO-1 pathway and regulating oral microbiota, PAT-W is expected to become a new natural drug for treating oral ulcers and improving patients' quality of life.

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.

Recombinant Oxidase from Armillaria tabescens as a Potential Tool for Aflatoxin B1 Degradation in Contaminated Cereal Grain.

Forage grain contamination with aflatoxin B1 (AFB1) is a global problem, so its detoxification with the aim of providing feed safety and cost-efficiency is still a relevant issue. AFB1 degradation by microbial enzymes is considered to be a promising detoxification approach. In this study, we modified an previously developed Pichia pastoris GS115 expression system using a chimeric signal peptide to obtain a new recombinant producer of extracellular AFB1 oxidase (AFO) from Armillaria tabescens (the yield of 0.3 g/L), purified AFO, and selected optimal conditions for AFO-induced AFB1 removal from model solutions. After a 72 h exposure of the AFB1 solution to AFO at pH 6.0 and 30 °C, 80% of the AFB1 was degraded. Treatments with AFO also significantly reduced the AFB1 content in wheat and corn grain inoculated with Aspergillus flavus. In grain samples contaminated with several dozen micrograms of AFB1 per kg, a 48 h exposure to AFO resulted in at least double the reduction in grain contamination compared to the control, while the same treatment of more significantly (~mg/kg) AFB1-polluted samples reduced their contamination by ~40%. These findings prove the potential of the tested AFO for cereal grain decontamination and suggest that additional studies to stabilize AFO and improve its AFB1-degrading efficacy are required.

Two distinct non-ribosomal peptide synthetase-independent siderophore synthetase gene clusters identified in Armillaria and other species in the Physalacriaceae.

Siderophores are important for ferric iron solubilization, sequestration, transportation, and storage, especially under iron-limiting conditions such as aerobic conditions at high pH. Siderophores are mainly produced by non-ribosomal peptide synthetase-dependent siderophore pathway, non-ribosomal peptide synthetase-independent siderophore synthetase pathway, or the hybrid non-ribosomal peptide synthetases/non-ribosomal peptide synthetases-independent siderophore pathway. Outcompeting or inhibition of plant pathogens, alteration of host defense mechanisms, and alteration of plant-fungal interactions have been associated with fungal siderophores. To understand these mechanisms in fungi, studies have been conducted on siderophore biosynthesis by ascomycetes with limited focus on the basidiomycetes. Armillaria includes several species that are pathogens of woody plants and trees important to agriculture, horticulture, and forestry. The aim of this study was to investigate the presence of non-ribosomal peptide synthetases-independent siderophore synthetase gene cluster(s) in genomes of Armillaria species using a comparative genomics approach. Iron-dependent growth and siderophore biosynthesis in strains of selected Armillaria spp. were also evaluated in vitro. Two distinct non-ribosomal peptide synthetases-independent siderophore synthetase gene clusters were identified in all the genomes. All non-ribosomal peptide synthetases-independent siderophore synthetase genes identified putatively encode Type A' non-ribosomal peptide synthetases-independent siderophore synthetases, most of which have IucA_IucC and FhuF-like transporter domains at their N- and C-terminals, respectively. The effect of iron on culture growth varied among the strains studied. Bioassays using the CAS assay on selected Armillaria spp. revealed in vitro siderophore biosynthesis by all strains irrespective of added FeCl3 concentration. This study highlights some of the tools that Armillaria species allocate to iron homeostasis. The information generated from this study may in future aid in developing molecular based methods to control these phytopathogens.

Critical review of the phytochemical profiles and health-promoting effects of the edible mushroom Armillaria mellea.

Research on the nutritional and medicinal properties of wild edible mushrooms has witnessed a significant surge in recent years. Among these mushrooms, Armillaria mellea (AM) stands out due to its abundant biologically active components. The presence of biological compounds in AM, including carbohydrates, sterols, fatty acids, sesquiterpenes, non-hallucinogenic indole compounds and adenosine derivatives, has been demonstrated in previous studies. Notably, specific bioactive substances isolated from AM, such as armillarikin, have exhibited promising anticancer effects. In vitro studies have elucidated the mechanisms behind these effects, further emphasizing the potential of AM in cancer treatment. Consequently, the objective of this study is to provide a comprehensive overview of the phytochemical profiles of AM while thoroughly investigating its therapeutic benefits. Moreover, this research has uncovered novel and effective treatments, including the utilization of ultrasonic disruption extraction in food processing. These findings highlight the potential of AM as a functional food with possible medical applications. By exploring AM's phytochemical composition and therapeutic effects, this study aims to contribute to a deeper understanding of its potential as a valuable natural resource.

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.

A practical approach to genome assembly and annotation of Basidiomycota using the example of Armillaria.

Technological advancements in genome sequencing, assembly and annotation platforms and algorithms that resulted in several genomic studies have created an opportunity to further our understanding of the biology of phytopathogens, including Armillaria species. Most Armillaria species are facultative necrotrophs that cause root- and stem-rot, usually on woody plants, significantly impacting agriculture and forestry worldwide. Genome sequencing, assembly and annotation in terms of samples used and methods applied in Armillaria genome projects are evaluated in this review. Infographic guidelines and a database of resources to facilitate future Armillaria genome projects were developed. Knowledge gained from genomic studies of Armillaria species is summarized and prospects for further research are provided. This guide can be applied to other diploid and dikaryotic fungal genomics.

Structural characterization, anti-tumor and immunomodulatory activity of intracellular polysaccharide from Armillaria luteo-virens.

Armillaria luteo-virens (A. luteo-virens) is a kind of edible fungus mainly exists in Qinghai-Tibet of China, but at present only very few studies focus on the bioactivities of its polysaccharides. This study aimed to purify and characterize the structure features of a novel intracellular polysaccharide (ALP-A) derived from A. luteo-virens and explore its potential anti-tumor and immunomodulatory activities. Through systematic separation and purification, we obtained a homogeneous ALP-A with an average molecular weight of 23693Da. Structural analysis indicated that ALP-A was mainly composed of glucose and mannose with a molar ratio of 6.02:1. The repeating unit of ALP-A was →4) -α-D-Glcp-(1→ backbone with α-Glcp-(1→ and α-Manp-(6→ side chains which branched at O-2 position. The anti-tumor assays in vivo suggested that ALP-A could effectively restrain S180 solid tumor growth, protect immune organs and promote the secretion of cytokines (IL2, IL6 and TNF-α) in serum. Besides, in vitro immunomodulatory assays indicated that ALP-A could improve proliferation, phagocytic capacity and raise the level of NO and cytokines in Raw264.7 cells. These results demonstrate that ALP-A which possess potential antitumor and immunomodulatory abilities can be developed as a new functional food.

Characterization of the transcriptional responses of Armillaria gallica 012m to GA3.

Gastrodia elata needs to establish a symbiotic relationship with Armillaria strains to obtain nutrients and energy. However, the signaling cross talk between G. elata and Armillaria strains is still unclear. During our experiment, we found that the vegetative mycelium of Armillaria gallica 012m grew significantly better in the media containing gibberellic acid (GA3) than the blank control group (BK). To explore the response mechanism, we performed an RNA-sequencing experiment to profile the transcriptome changes of A. gallica 012m cultured in the medium with exogenous GA3. The transcriptome-guided differential expression genes (DEGs) analysis of GA3 and BK showed that a total of 1309 genes were differentially expressed, including 361 upregulated genes and 948 downregulated genes. Some of those DEGs correlated with the biological process, including positive regulation of chromosome segregation, mitotic metaphase/anaphase transition, attachment of mitotic spindle microtubules to kinetochore, mitotic cytokinesis, and nuclear division. These analyses explained that GA3 actively promoted the growth of A. gallica to some extent. Further analysis of protein domain features showed that the deduced polypeptide contained 41 candidate genes of GA receptor, and 27 of them were expressed in our samples. We speculate that GA receptors exist in A. gallica 012m. Comparative studies of proteins showed that the postulated GA receptor domains of A. gallica 012m have a higher homologous correlation with fungi than others based on cluster analysis.

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria.

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have affected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens.

Dual RNA-Seq Profiling Unveils Mycoparasitic Activities of Trichoderma atroviride against Haploid Armillaria ostoyae in Antagonistic Interaction Assays.

Armillaria ostoyae, a species among the destructive forest pathogens from the genus Armillaria, causes root rot disease on woody plants worldwide. Efficient control measures to limit the growth and impact of this severe underground pathogen are under investigation. In a previous study, a new soilborne fungal isolate, Trichoderma atroviride SZMC 24276 (TA), exhibited high antagonistic efficacy, which suggested that it could be utilized as a biocontrol agent. The dual culture assay results indicated that the haploid A. ostoyae-derivative SZMC 23085 (AO) (C18/9) is highly susceptible to the mycelial invasion of TA. In the present study, we analyzed the transcriptome of AO and that of TA in in vitro dual culture assays to test the molecular arsenal of Trichoderma antagonism and the defense mechanisms of Armillaria. We conducted time-course analysis and functional annotation and analyzed enriched pathways and differentially expressed genes including biocontrol-related candidate genes from TA and defense-related candidate genes from AO. The results indicated that TA deployed several biocontrol mechanisms when confronted with AO. In response, AO initiated multiple defense mechanisms to protect against the fungal attack. To our knowledge, the present study offers the first transcriptome analysis of a biocontrol fungus attacking AO. Overall, this study provides insights that aid the further exploration of plant pathogen-biocontrol agent interaction mechanisms. IMPORTANCE Armillaria species can survive for decades in the soil on dead woody debris, develop rapidly under favorable conditions, and harmfully infect newly planted forests. Our previous study found Trichoderma atroviride to be highly effective in controlling Armillaria growth; therefore, our current work explored the molecular mechanisms that might play a key role in Trichoderma-Armillaria interactions. Direct confrontation assays combined with time course-based dual transcriptome analysis provided a reliable system for uncovering the interactive molecular dynamics between the fungal plant pathogen and its mycoparasitic partner. Furthermore, using a haploid Armillaria isolate allowed us to survey the deadly prey-invading activities of the mycoparasite and the ultimate defensive strategies of its prey. Our current study provides detailed insights into the essential genes and mechanisms involved in Armillaria defense against Trichoderma and the genes potentially involved in the efficiency of Trichoderma to control Armillaria. In addition, using a sensitive haploid Armillaria strain (C18/9), with its complete genome data already available, also offers the opportunity to test possible variable molecular responses of Armillaria ostoyae toward diverse Trichoderma isolates with various biocontrol abilities. Initial molecular tests of the dual interactions may soon help to develop a targeted biocontrol intervention with mycoparasites against plant pathogens.

Whole genome sequencing and analysis of Armillaria gallica Jzi34 symbiotic with Gastrodia elata.

BACKGROUND: Armillaria species are plant pathogens, but a few Armillaria species can establish a symbiotic relationship with Gastrodia elata, a rootless and leafless orchid, that is used as a Chinese herbal medicine. Armillaria is a nutrient source for the growth of G. elata. However, there are few reports on the molecular mechanism of symbiosis between Armillaria species and G. elata. The genome sequencing and analysis of Armillaria symbiotic with G. elata would provide genomic information for further studying the molecular mechanism of symbiosis. RESULTS: The de novo genome assembly was performed with the PacBio Sequel platform and Illumina NovaSeq PE150 for the A. gallica Jzi34 strain, which was symbiotic with G. elata. Its genome assembly contained ~ 79.9 Mbp and consisted of 60 contigs with an N50 of 2,535,910 bp. There were only 4.1% repetitive sequences in the genome assembly. Functional annotation analysis revealed a total of 16,280 protein coding genes. Compared with the other five genomes of Armillaria, the carbohydrate enzyme gene family of the genome was significantly contracted, while it had the largest set of glycosyl transferase (GT) genes. It also had an expansion of auxiliary activity enzymes AA3-2 gene subfamily and cytochrome P450 genes. The synteny analysis result of P450 genes reveals that the evolutionary relationship of P450 proteins between A. gallica Jzi34 and other four Armillaria was complex. CONCLUSIONS: These characteristics may be beneficial for establishing a symbiotic relationship with G. elata. These results explore the characteristics of A. gallica Jzi34 from a genomic perspective and provide an important genomic resource for further detailed study of Armillaria. This will help to further study the symbiotic mechanism between A. gallica and G. elata.

Study of anti-fatigue activity of polysaccharide from fruiting bodies of Armillaria gallica.

Fatigue is a common physiological response that is closely related to energy metabolism. Polysaccharides, as excellent dietary supplements, have been proven to have a variety of pharmacological activities. In this study, A 23.007 kDa polysaccharide from Armillaria gallica (AGP) was purified and performed structural characterization, including analysis of homogeneity, molecular weight and monosaccharide composition. Methylation analysis is used to analyze the glycosidic bond composition of AGP. The mouse model of acute fatigue was used to evaluate the anti-fatigue effect of AGP. AGP-treatment improved exercise endurance in mice and reduced fatigue symptoms caused by acute exercise. AGP regulated the levels of adenosine triphosphate, lactic acid, blood urea nitrogen and lactate dehydrogenase, muscle glycogen and liver glycogen of acute fatigue mice. AGP affected the composition of intestinal microbiota, the changes of some intestinal microorganisms are correlated with fatigue and oxidative stress indicators. Meanwhile, AGP reduced oxidative stress levels, increased antioxidant enzyme activity and regulated the AMP-dependent protein kinase/nuclear factor erythroid 2-related factor 2 signaling pathway. AGP exerted an anti-fatigue effect through modulation of oxidative stress, which is related to intestinal microbiota.

Chemical Characterization and Bioactive Properties of the Edible and Medicinal Honey Mushroom Armillaria mellea (Agaricomycetes) from Serbia.

Honey mushroom, Armillaria mellea, is an edible and medicinal lignicolous basidiomycete. In this study, we investigated the chemical composition and bioactive properties of its methanolic and acetonic extracts. The chemical characterization of extracts was done with the HPLC-DAD-MS/MS method. The results showed that potassium was the most abundant mineral; chlorogenic acid was the most abundant polyphenol; malic acid was the most abundant organic acid; and among carbohydrates, the most abundant were sorbitol, glucose, fructose, and saccharose. Antioxidative activity was assessed by DPPH (IC50 of the methanolic extract was 608.32 µg/mL and of the acetonic extract 595.71 µg/mL) and reducing power assays (the results ranged between 0.034 and 0.102 µg/mL). Total phenolic content was determined as gallic acid equivalent (methanolic extract: 4.74 mg GAE/g; acetonic extract: 5.68 mg GAE/g). The microdilution assay was used to evaluate the antimicrobial activity of the extracts, and the results ranged from 1.25 to 20 mg/mL. The antidiabetic effect of the extracts was tested by the α-amylase (the results ranged from 34.90% to 41.98%) and α-glucosidase assays (the results were in the range of 0.55-2.79%). The neuroprotective activity was explored by the acetylcholinesterase inhibition assay (the results were in the range of 1.94-7.76%). The microtetrazolium assay was used to investigate the cytotoxic activity of the extracts (the IC50 values ranged from 212.06 to > 400 µg/mL). Although some results suggest that some activities of the extracts are relatively moderate, the honey mushroom can still be considered an excellent source of food and bioactive compounds with medicinal value.