Determination of the Absolute Configuration of Secondary Alcohols in a Compound Mixture via the Application of Competing Enantioselective Acylation Coupled with LC/MS Analysis.

The determination of natural product stereochemistry plays a significant role in drug discovery and development. Understanding the stereochemistry of natural products is essential for predicting and optimizing their interactions with biological targets, which, in turn, influences their therapeutic efficacy, safety, and overall impact on living organisms. Here, we present the first application of competitive enantioselective acylation (CEA) reactions in conjunction with LC/MS analysis for determining the absolute configuration of secondary alcohols in natural products which were purified as a mixture. This approach utilizes the enantiomeric pair of HBTM (homobenzotetramisole) catalysts, demonstrating sufficient kinetic resolution for the acylation of secondary alcohols. The rapid reaction kinetics were quantitatively estimated with LC/MS analysis as the characterization technique for the enantioselective transformations. Our study has expanded the application of the CEA reaction coupled with LC/MS analysis to mixtures. Utilizing LC/MS analysis, the CEA reaction offers a sensitive and simple method for stereochemistry determination. Additionally, the application of the CEA reaction is cost/time-effective since only small quantities of substrates and a short reaction time are required for characterizing the absolute configuration of secondary alcohols in natural products compared to other conventional methods.

Potential Antiviral Effect of Korean Forest Wild Mushrooms against Feline Coronavirus (FCoV).

Coronaviruses (CoV) are among the major viruses that cause common cold in humans. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a high-risk human pathogen that derived from bat coronaviruses, although several other animals serve as CoV hosts, contributing to human infection. As the human activity area expanded, viruses previously prevalent only in animals mutated and became threats to humans as well, leading to worldwide epidemics. Therefore, controlling CoV infections in animals is essential to prevent CoV-related human infections. Feline coronavirus (FCoV) could be reportedly used as an alternative model for SARS-CoV-2. Traditionally, mushrooms are not only foods but are also consumed to prevent diseases. Importantly, certain edible and medicinal mushrooms display antibacterial and antiviral effects against respiratory pathogens; therefore, they could be tested as potential coronavirus treatment agents. In this study, we investigated if wild forest mushrooms with various reported physiological activities could exhibit an antiviral activity against CoV, using FCoV as a SARS-CoV-2 model infecting Crandell Rees feline kidney cells. We measured the antiviral activity of 11 wild mushrooms overall and our results demonstrated that Pleurotus ostreatus and Phallus luteus displayed the highest antiviral efficacy of 55.33%, followed by Tricholoma bakamatsutake at 43.77%. Grifola frondosa, Morchella esculenta, and Sarcodon imbricatus exhibited mild efficacy of 29.21%. We also tested Amanita caesareoides, Marasmius siccus, Pachyma hoelen, Phallus rubrovolvata, and Sparassis latifolia but could not detect any antiviral activity in their case. Our study confirms that wild forest mushrooms could be used as potential functional foods or pharmacological materials against coronavirus.

New autophagy-modulating lanostane-type triterpenoids from a hallucinogenic poisonous mushroom Gymnopilus orientispectabilis.

Gymnopilus orientispectabilis, also known as "big laughter mushroom," is a hallucinogenic poisonous mushroom that causes excessive laughter upon ingestion. From the fruiting bodies of G. orientispectabilis, eight lanostane-type triterpenoids (1-8), including seven novel compounds: gymnojunols A-G (2-8), were isolated. The chemical structures of these new compounds (2-8) were determined by analyzing their 1D and 2D NMR spectra and HR-EISMS, and their absolute configurations were unambiguously assigned by quantum chemical ECD calculations and a computational method coupled with a statistical procedure (DP4+). Upon evaluating autophagic activity, compounds 2, 6, and 7 increased LC3B-II levels in HeLa cells to a similar extent as bafilomycin, an autophagy inhibitor. In contrast, compound 8 decreased the levels of both LC3B-I and LC3B-II, and a similar effect was observed following treatment with rapamycin, an autophagy inducer. Our findings provide experimental evidence for new potential autophagy modulators in the hallucinogenic poisonous mushroom G. orientispectabilis.

Cloning and Expression Analysis of Bioluminescence Genes in Omphalotus guepiniiformis Reveal Stress-Dependent Regulation of Bioluminescence.

Bioluminescence is a type of chemiluminescence that arises from a luciferase-catalyzed oxidation reaction of luciferin. Molecular biology and comparative genomics have recently elucidated the genes involved in fungal bioluminescence and the evolutionary history of their clusters. However, most studies on fungal bioluminescence have been limited to observing the changes in light intensity under various conditions. To understand the molecular basis of bioluminescent responses in Omphalotus guepiniiformis under different environmental conditions, we cloned and sequenced the genes of hispidin synthase, hispidin-3-hydroxylase, and luciferase enzymes, which are pivotal in the fungal bioluminescence pathway. Each gene showed high sequence similarity to that of other luminous fungal species. Furthermore, we investigated their transcriptional changes in response to abiotic stresses. Wound stress enhanced the bioluminescence intensity by increasing the expression of bioluminescence pathway genes, while temperature stress suppressed the bioluminescence intensity via the non-transcriptional pathway. Our data suggested that O. guepiniiformis regulates bioluminescence to respond differentially to specific environmental stresses. To our knowledge, this is the first study on fungal bioluminescence at the gene expression level. Further studies are required to address the biological and ecological meaning of different bioluminescence responses in changing environments, and O. quepiniiformis could be a potential model species.

N-Hydroxy-Phe-Phe, a new dipeptide, and cytotoxic macrocyclic trichothecenes from the lethal toxic mushroom Podostroma cornu-damae.

Podostroma cornu-damae, commonly referred to as the red deer's horn mushroom due to its distinct resemblance to the antlers of a deer, is a lethal toxic mushroom that causes vomiting, dehydration, diarrhea, disturbance of consciousness, and even death. In continuation of our research aiming to investigate the novel structural and/or biological principles present in Korean wild mushrooms, a new N-hydroxyphenylalanine-phenylalanine dipeptide, N-hydroxy-Phe-Phe (1), and three known macrocyclic trichothecenes, satratoxin H (2), 12'-episatratoxin H (3), and roridin F (4), were isolated from the MeOH extract of a plate culture of the poisonous mushroom P. cornu-damae. The chemical structure of the new dipeptide (1) was determined by analyzing 1D and 2D NMR spectra and high-resolution (HR)-electrospray ionization mass spectroscopy (ESIMS), along with a computational method combined with a statistical procedure (DP4+), and its absolute configuration was unambiguously assigned by quantum chemical ECD calculations. To the best of our knowledge, compound 1 is the first dipeptide found in P. cornu-damae. Upon evaluating the cytotoxicity of compounds 1-4 against four human-derived cancer cell lines namely SK-OV-3, SK-MEL-2, A549, and HCT15, 12'-episatratoxin H (3) displayed potent cytotoxic effects toward all four cell lines tested, with IC50 values ranging from 0.7 to 2.8 nM, which was found to be stronger than that of doxorubicin. Satratoxin H (2) also demonstrated moderate cytotoxic potency against all four cell lines, with IC50 values ranging from 1.93 to 4.22 µM. Our findings provide experimental data supporting the potential of the poisonous mushroom P. cornu-damae as a source of anticancer agents.

N,N-Dimethyl-anthranilic Acid from Calvatia nipponica Mushroom Fruiting Bodies Induces Apoptotic Effects on MDA-MB-231 Human Breast Cancer Cells.

Breast cancer ranks among the most prevalent malignancies affecting women worldwide, and apoptosis-targeting drugs are attractive candidates for the treatment of cancer. In the current study, we investigated the in vitro cytotoxicity of the mushroom Calvatia nipponica in human breast cancer cells (MDA-MB-231), identified potential antitumor compounds through bioactivity-guided isolation, and elucidated the antitumor, pro-apoptotic molecular mechanisms of the identified bioactive compounds. C. nipponica is edible when young, and it has been used as a food source as well as a traditional medicine in wound dressings. However, only a limited number of studies have reported its chemical composition and biological activities. In the screening test, the methanol extract of C. nipponica fruiting bodies exhibited cytotoxicity against MDA-MB-231 cells. Bioactivity-guided fractionation of the methanol (MeOH) extract and chemical investigation of the active fractions resulted in the isolation of fourteen compounds (1-14), including six alkaloids (1-3, 5, 7, and 8), two phenolic compounds (4 and 6), one fatty acid (9), and five steroids (10-14). The structures of the isolated compounds were determined using NMR spectroscopic methods, liquid chromatography-mass spectrometry, and comparison of data with previously reported values. The isolated compounds (1-14) were tested for cytotoxicity against MDA-MB-231 cells, where compound 1, i.e., N,N-dimethyl-anthranilic acid, exhibited the most significant cytotoxicity against MDA-MB-231 cells, with an IC50 value of 90.28 ± 4.23 µM and apoptotic cell death of 56.01% ± 2.64% at 100 µM. Treatment with compound 1 resulted in an upregulation of protein levels, including cleaved caspase-8, cleaved poly (ADP-ribose) polymerase, Bcl-2-associated X protein (Bax), cleaved caspase-3, cleaved caspase-9, Bad, and Cytochrome c, but decreased the levels of B-cell lymphoma 2 (Bcl-2). Overall, these results indicate that N,N-dimethyl-anthranilic acid (1) may have anti-breast cancer activity and is probably involved in the induction of apoptosis mediated by extrinsic and intrinsic signaling pathways.

Inhibition of Osteoclast Differentiation and Promotion of Osteogenic Formation by Wolfiporia extensa Mycelium.

Osteoporosis, Greek for "porous bone," is a bone disease characterized by a decrease in bone strength, microarchitectural changes in the bone tissues, and an increased risk of fracture. An imbalance of bone resorption and bone formation may lead to chronic metabolic diseases such as osteoporosis. Wolfiporia extensa, known as "Bokryung" in Korea, is a fungus belonging to the family Polyporaceae and has been used as a therapeutic food against various diseases. Medicinal mushrooms, mycelium and fungi, possess approximately 130 medicinal functions, including antitumor, immunomodulating, antibacterial, hepatoprotective, and antidiabetic effects, and are therefore used to improve human health. In this study, we used osteoclast and osteoblast cell cultures treated with Wolfiporia extensa mycelium water extract (WEMWE) and investigated the effect of the fungus on bone homeostasis. Subsequently, we assessed its capacity to modulate both osteoblast and osteoclast differentiation by performing osteogenic and anti-osteoclastogenic activity assays. We observed that WEMWE increased BMP-2-stimulated osteogenesis by inducing Smad-Runx2 signal pathway axis. In addition, we found that WEMWE decreased RANKL-induced osteoclastogenesis by blocking c-Fos/NFATc1 via the inhibition of ERK and JNK phosphorylation. Our results show that WEMWE can prevent and treat bone metabolic diseases, including osteoporosis, by a biphasic activity that sustains bone homeostasis. Therefore, we suggest that WEMWE can be used as a preventive and therapeutic drug.

De novo genome assembly of the bioluminescent mushroom Omphalotus guepiniiformis reveals an Omphalotus-specific lineage of the luciferase gene block.

Omphalotus guepiniiformis, a bioluminescent mushroom species, is a source of the potentially valuable anticancer chemical. To provide genome information, we de novo assembled the high-quality O. guepiniiformis genome using two Next-Generation sequencing techniques, PacBio and Illumina sequencing. Our draft O. guepiniiformis genome comprises 42.5 Mbp of sequence with only 80 contigs and an N50 sequence length of over 1 Mbp. There were 15,554 predicted coding genes, and 7693 genes were functionally annotated with Gene Ontology terms. We performed a genomic study focusing on mushroom bioluminescent pathway cluster genes by comparing 17 luminescent and 23 non-luminescent Agaricales species belonging to 23 genera. Synteny analysis of genomic regions near the luminescent pathway cluster genes inferred that the Omphalotus lineage was genus-specific. In summary, our de novo assembled O. guepiniiformis genome provides significant biological insights into this organism, including the evolution of the luciferase gene block, and forms the basis for future analyses.

Meyeroguilline E, a New Isoindolinone Alkaloid from the Poisonous Mushroom Chlorophyllum molybdites, and Identification of Compounds with Multidrug Resistance (MDR) Reversal Activities.

Three isoindolinone alkaloids (1-3), including one new isoindolinone-type alkaloid, meyeroguilline E (1), and six other known compounds (4-9) were isolated from the poisonous mushroom Chlorophyllum molybdites (Agaricaceae). The structure of the new compound was determined using extensive spectroscopic analyses via one-dimensional (1D) and two-dimensional (2D) NMR data interpretation and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). To the best of our knowledge, compound 1 is the first example of a natural isoindolinone with a butanoic acid moiety, and this study is the first to detect the other known compounds (2-9) in C. molybdites. The isolated compounds (1-9) were examined for their multidrug resistance (MDR) reversal activity against MES-SA, MES-SA/DX5, HCT15, and HCT15/CL02 human cancer cells. Based on the results, 20 µM of compounds 3 and 6 slightly potentiated paclitaxel (TAX)-induced cytotoxicity in MES-SA/DX5, HCT15, and HCT15/CL02 cells; however, the compounds had no effect on the cytotoxicity against MES-SA and nonMDR cells.

Identification of Antibacterial Sterols from Korean Wild Mushroom Daedaleopsis confragosa via Bioactivity- and LC-MS/MS Profile-Guided Fractionation.

As part of an ongoing natural product chemical research for the discovery of bioactive secondary metabolites with novel structures, wild fruiting bodies of Daedaleopsis confragosa were collected and subjected to chemical and biological analyses. We subjected the fractions derived from the methanol extract of the fruiting bodies of D. confragosa to bioactivity-guided fractionation because the methanol extract of D. confragosa showed antibacterial activity against Helicobacter pylori strain 51, according to our bioactivity screening. The n-hexane and dichloromethane fractions showed moderate to weak antibacterial activity against H. pylori strain 51, and the active fractions were analyzed for the isolation of antibacterial compounds. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the n-hexane fraction contains several compounds which are absent in the other fractions, so the fraction was prioritized for further fractionation. Through chemical analysis of the active n-hexane and dichloromethane fractions, we isolated five ergosterol derivatives (1-5), and their chemical structures were determined to be demethylincisterol A3 (1), (20S,22E,24R)-ergosta-7,22-dien-3ß,5α,6ß-triol (2), (24S)-ergosta-7-ene-3ß,5α,6ß-triol (3), 5α,6α-epoxy-(22E,24R)-ergosta-7,22-dien-3ß-ol (4), and 5α,6α-epoxy-(24R)-ergosta-7-en-3ß-ol (5) by NMR spectroscopic analysis. This is the first report on the presence of ergosterol derivatives (1-5) in D. confragosa. Compound 1 showed the most potent anti-H. pylori activity with 33.9% inhibition, rendering it more potent than quercetin, a positive control. Compound 3 showed inhibitory activity comparable to that of quercetin. Distribution analysis of compound 1 revealed a wide presence of compound 1 in the kingdom Fungi. These findings indicate that demethylincisterol A3 (1) is a natural antibiotic that may be used in the development of novel antibiotics against H. pylori.

Two New Fatty Acid Derivatives, Omphalotols A and B and Anti-Helicobacter pylori Fatty Acid Derivatives from Poisonous Mushroom Omphalotus japonicus.

As part of ongoing systematic research into the discovery of bioactive secondary metabolites with novel structures from Korean wild mushrooms, we investigated secondary metabolites from a poisonous mushroom, Omphalotus japonicus (Kawam.) Kirchm. & O. K. Mill. belonging to the family Marasmiaceae, which causes nausea and vomiting after consumption. The methanolic extract of O. japonicus fruiting bodies was subjected to the fractionation by solvent partition, and the CH2Cl2 fraction was analyzed for the isolation of bioactive compounds, aided by an untargeted liquid chromatography mass spectrometry (LC-MS)-based analysis. Through chemical analysis, five fatty acid derivatives (1-5), including two new fatty acid derivatives, omphalotols A and B (1 and 2), were isolated from the CH2Cl2 fraction, and the chemical structures of the new compounds were determined using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and high resolution electrospray ionization mass spectrometry (HR-ESIMS), as well as fragmentation patterns in MS/MS data and chemical reactions followed by the application of Snatzke's method and competing enantioselective acylation (CEA). In the anti-Helicobacter pylori activity test, compound 1 showed moderate antibacterial activity against H. pylori strain 51 with 27.4% inhibition, comparable to that of quercetin as a positive control. Specifically, compound 3 exhibited the most significant antibacterial activity against H. pylori strain 51, with MIC50 and MIC90 values of 9 and 20 µM, respectively, which is stronger inhibitory activity than that of another positive control, metronidazole (MIC50 = 17 µM and MIC90 = 46 µM). These findings suggested the experimental evidence that the compound 3, an α,ß-unsaturated ketone derivative, could be used as a moiety in the development of novel antibiotics against H. pylori.

First Chemical Investigation of Korean Wild Mushroom, Amanita hemibapha subsp. javanica and the Identification of Anti-Helicobacter pylori Compounds.

Amanita hemibapha subsp. javanica (Amanitaceae) is an edible Korean wild mushroom. A. hemibapha subsp. javanica is often confused with A. subjunquillea, known as the East Asian death cap, which is potentially fatal when ingested. This study aimed to conduct the first chemical investigation of A. hemibapha subsp. javanica, which resulted in the isolation of seven fatty acid derivatives (1-7) and three steroids (8-10) from the MeOH extract of its fruiting bodies, and their structures were determined by comparing their NMR spectroscopic data with those previously reported, along with the data from LC/MS. Compound 1 was reported previously without the identification of its absolute configuration; its structure, including the absolute configuration was confirmed for the first time, in this study, by using 1H NMR and its fragmentation patterns in MS/MS data, and LC/MS analysis. A recently developed method using competing enantioselective acylation (CEA) coupled with LC/MS analysis was applied for determining the absolute configuration of compound 1, which revealed the 11S-configuration. In the anti-Helicobacter pylori activity test, compound 3 showed antibacterial activity against H. pylori strain 51 with 38.0% inhibition, comparable to that of quercetin (34.4% inhibition) as a positive control. Specifically, compound 4 displayed the most potent antibacterial activity against H. pylori strain 51 with 80.5% inhibition at the final concentration of 100 µm with a MIC50 value of 72 µm. These findings suggested that the active compound 4 is a natural antibiotic that may be used in the development of novel antibiotics against H. pylori. In addition, the first chemical investigation of A. hemibapha subsp. javanica revealed that this mushroom can serve as a promising natural source for the bioactive natural products.

Pulveraven A from the fruiting bodies of Pulveroboletus ravenelii induces apoptosis in breast cancer cell via extrinsic apoptotic signaling pathway.

Pulveroboletus ravenelii (Beck. et Curt.) Murr. (Boletaceae), commonly known as Ravenel's bolete, is an edible and medicinal mushroom, and is also used for preparing mushroom-based dyes. As part of a continuing project to discover the bioactive natural products from wild mushrooms, we analyzed the methanol (MeOH) extract of P. ravenelii to identify metabolites with the anticancer activity. Chemical analysis of the MeOH extract combined with liquid chromatography-mass spectrometry (LC-MS) analysis led to the isolation of a phenolic compound, pulveraven A (PA), whose chemical structure was determined using a combination of 1D and 2D NMR and LC-MS analysis. In the present study, we investigated the cytotoxicity and anticancer mechanisms of pulveraven A using human breast cancer (MCF-7) cells, and demonstrated that it reduced cell viability of MCF-7 cells below 50% (71.74 ± 3.61 µM). Annexin V Alexa Fluor 488 binding assay and western blot results revealed that pulveraven A induced apoptotic cell death via the extrinsic apoptosis pathway, as indicated by the activation of initiator caspase-8 and executioner caspase-7. Furthermore, it was accompanied by an increase in the Bax/Bcl-2 ratio. These results suggest that pulveraven A induces apoptosis in breast cancer cells via the extrinsic apoptotic signaling pathway.

Ergopyrone, a Styrylpyrone-Fused Steroid with a Hexacyclic 6/5/6/6/6/5 Skeleton from a Mushroom Gymnopilus orientispectabilis.

A styrylpyrone-fused ergosterol derivative, ergopyrone (1), was isolated and structurally characterized from a mushroom, Gymnopilus orientispectabilis, along with five biosynthetically related metabolites (2-6). Compound 1 features an unprecedented hexacyclic 6/5/6/6/6/5 skeleton that would be formed from ergosterol and styrylpyrone precursors via [3 + 2] cycloaddition. The chemical structure of 1 was elucidated by conventional spectroscopic and spectrometric data analysis coupled with computational methods including DP4+ probability and ECD simulation and an NOE/ROE-based interproton distance measurement technique via peak amplitude normalization for the improved cross-relaxation (PANIC) method. Plausible biosynthetic pathways of 1-6 are proposed, and compound 6 significantly regulated lipid metabolism in adipocytes through the upregulation of the mRNA expression of Adipsin, Fabp4, SREBP1, and ATGL.

Bioactivity-based analysis and chemical characterization of cytotoxic compounds from a poisonous mushroom, Amanita spissacea, in human lung cancer cells in vitro.

As part of our systematic study on Korean toxic mushrooms, bioactivity-guided fractionation of the MeOH extract of Amanita spissacea (Amanitaceae) fruiting bodies and chemical investigation of its cytotoxic fractions led to the isolation of (9E)-8-oxo-9-octadecenoic acid (1), (10E)-9-oxo-10-octadecenoic acid (2), (9E)-8-oxo-9-octadecenoate methyl ester (3), (9Z)-9-octadecenoate-(2'S)-2',3'-dihydroxypropyl ester (4), (9Z)-9-octadecenoic acid (5), and palmitic acid (6). The structures of the isolates were elucidated by NMR spectroscopic analysis and LC/MS analysis. Among the isolated compounds, compounds 1 and 2 exhibited the most potent cytotoxic activity in all human lung cancer cell lines examined, with IC50 values ranging from 255.7 to 321.0 µM and 250.2 to 322.5 µM, respectively. The cytotoxicity of these compounds was also found to be mediated by apoptosis associated with caspase-3 activation. These findings provide experimental evidence suggesting the potential of A. spissacea as a promising natural source for the discovery of novel anticancer drug candidates.

Marasmioid and Gymnopoid Fungi of the Republic of Korea. 8. Gymnopus Section Levipedes.

Collections of Gymnopus sect. Levipedes from the Republic of Korea have been studied. Two new species, Gymnopus dryophiloides and G. brunneodiscus , are described based on their macro- and micromorphological and phylogenetic characteristics. Three other species, referred to as Gymnopus spp. 1, 2, and 3, are distinguished as separate taxa without formal descriptions. Taxonomic and phylogenetic positions of all taxa have been inferred and confirmed by analyses of ITS and LSU sequence data. Their detailed descriptions, illustrations and an identification key are provided.

Calvatianone, a Sterol Possessing a 6/5/6/5-Fused Ring System with a Contracted Tetrahydrofuran B-Ring, from the Fruiting Bodies of Calvatia nipponica.

Calvatia nipponica is an extremely rare mushroom with a limited number of studies on its chemical components and biological activities published. Here we report the isolation of a novel sterol, calvatianone (1), possessing a 6/5/6/5-fused ring system with a contracted tetrahydrofuran B-ring, and four known steroids (2-5) from the fruiting bodies of C. nipponica. The structure of calvatianone including its absolute configuration was determined by NMR spectroscopic analyses, HR-ESIMS, gauge-including atomic orbital NMR chemical shift calculations, and ECD calculations. Ergosterol peroxide (3) and cyathisterol (4) suppressed the cell viability increase induced by 17ß-estradiol in MCF-7 breast cancer cell lines, suggesting a possible approach for these compounds to serve as ERα antagonists.

Ergostane-Type Steroids from Korean Wild Mushroom Xerula furfuracea that Control Adipocyte and Osteoblast Differentiation.

As part of our current work to discover structurally and/or biologically novel compounds from Korean wild mushrooms, we isolated five ergostane-type steroids (1-5) from the fruiting bodies of Xerula furfuracea via repeated column chromatographic separations and HPLC purification. The chemical structures of the isolated steroids were shown to be (22E,24R)-24-methylcholesta-4,22- diene-3,6-dione (1), ergosta-7,22-diene-3ß,5α,6ß-triol (2), ergosta-7,22-diene-3ß,5α,6ß,9α-tetraol (3), (22E,24R)-5α,8α-epidioxyergosta-6,22-diene-3ß-ol-3-O-ß-D-glucopyranoside (4), and (22E,24R)-5α,8α-epidioxyergosta-6,9,22-triene-3ß-ol-3-O-ß-D-glucopyranoside (5) based on comparison of the data regarding their spectroscopic and physical properties with those of previous studies. Notably, this is the first report on the presence of the identified steroids (1-5) in this mushroom. We tested compounds 1-5 to determine their effects on adipogenesis and osteogenesis in the mouse mesenchymal stem cell line C3H10T1/2 and found that compounds 4 and 5 suppressed the differentiation of stem cells into adipocytes. Notably, in addition to its suppressive effect on adipogenesis, compound 5 was also shown to promote the osteogenic differentiation of stem cells. These findings demonstrate that the bioactive compounds isolated might be effective for the treatment of menopause-associated syndromes, such as osteoporosis and obesity, as the isolated compounds were shown to suppress adipogenesis and/or promote osteogenesis of stem cells.

Phallac acids A and B, new sesquiterpenes from the fruiting bodies of Phallus luteus.

Phallus luteus (Phallaceae), previously known as Dictyophora indusiata, is an edible and medicinal mushroom. As part of a continuing project to discover structurally and/or biologically novel natural products from wild mushrooms, we aimed to perform a chemical investigation of the methanol extract of P. luteus combined with a liquid chromatography-mass spectrometry-guided analysis coupled to an in-house UV spectral library. Two new sesquiterpenes, phallac acids A (1) and B (2), were isolated and determined. The chemical structure of the new natural products was unambiguously determined using a combination of 1D and 2D NMR and high-resolution electrospray ionization mass spectrometry data. To our knowledge, this is the first study to report linear sesquiterpene carboxylic acids from P. luteus. The new compounds were evaluated for α-glucosidase inhibitory activities where phallac acid B (2) showed α-glucosidase inhibitory potential (IC50 value of 94.89 ± 5.57 µM) compared with the standard acarbose (IC50 value of 26.23 ± 1.31 µM).

Cumulative Effects of Constituents from the Mushroom Calvatia nipponica on the Contractility of Penile Corpus Cavernosum Smooth Muscle.

Calvatia nipponica, a puffball mushroom (Agaricaceae), is thought to be an aphrodisiac, as this mushroom is traditionally known to improve sexual function in males. As part of the systematic study to determine the bioactive secondary metabolites from C. nipponica responsible for aphrodisiac effects, chemical analysis of methanol (MeOH) extracts of the fruiting bodies of C. nipponica resulted in the isolation of two major compounds: N,N-dimethyl-anthranilic acid (1) and (7Z,10Z)-7,10-octadecadienoic acid methyl ester (2). Compounds 1 and 2 were evaluated for cumulative dose-dependent relaxation responses to precontracted penile corpus smooth muscle (PCCSM). Results show that compounds 1 and 2 exhibited a maximum relaxation effect of 20.33 ± 2.18% and 24.63 ± 3.60%, respectively. These findings indicate that compounds 1 and 2, major components of C. nipponica, could potentially be used to treat erectile dysfunction, functioning as natural aphrodisiacs.