Cyclic Peptides from the Opportunistic Pathogen Basidiobolus meristosporus.

Three new cyclic peptides, meristosporins A, B, and C (1-3), one of which features an unusual amino acid, were isolated from the opportunistic pathogen Basidiobolus meristosporus and identified by 1D, 2D NMR, MS/MS, and Marfey's analysis. The biosynthetic pathway of the hexapeptide meristosporin A (1) was deduced based on nonribosomal peptide synthetase gene clusters analysis. Compounds 1 and 2 showed cytotoxicity to RAW264.7 and 293T cells, respectively. These compounds may be involved in the fungal injury caused to human cells.

Cyclic pentapeptides with anti-inflammatory, cytotoxic or α-glucosidase inhibitory activities from Basidiobolus meristosporus.

Basidiobolus meristosporus is an opportunistic pathogen of mammals with unique habitats, but its metabolites have not been extensively studied. Through semi-preparative HPLC, nine undescribed cyclic pentapeptides were isolated from mycelia of B. meristosporus RCEF4516. The structure of the compounds 1-9 were identified with MS/MS and NMR data and designated as basidiosin D-L respectively. The absolute configurations were determined according to the advanced Marfey's method after compound hydrolysis. Bioactivity testing showed that compounds 1, 2, 3, 4 and 8 decreased NO production in LPS-activated RAW264.7 cells in a concentration-dependent manner. The nine compounds showed cytotoxicity against RAW264.7, 293 T and HepG2 cells. All the compounds except compound 7 showed stronger inhibitory effects on α-glucosidase than acarbose.

Metabolites and novel compounds with anti-microbial or antiaging activities from Cordyceps fumosorosea.

High-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analysis revealed that there are 20 main components in spores and mycelia extract of Cordyceps fumosorosea strain RCEF 6672 including mannitol (1), uridine (2), adenine (3). N6-(2-hydroxyethyl)-adenosine (4). N6-(2-hydroxyethylacetate)-adenosine (5), fumosoroseanoside A (6) and B (7), ovalicin-4α-alcohol (8), 1-linoleoyl-sn-glycero-3-phosphocholine (9) and its isomer (10), fumosoroseain A (11) and its isomer (12), 5 non-ribosomal peptides (13 to 17) and 3 fatty acids (18 to 20). The compounds 5, 6, 7, 9 and 11 were prepared with preparative and semi-preparative HPLC and identified with 1D and 2D NMR. Compounds 4 and 5 were the first time identified from C. fumosorosea. Compounds 6, 7 and 11 are novel compounds. Compounds 6 and 7 showed antibacterial and antifungal activities, and 11 showed antiaging activity. All the secondary metabolites (4 to 8 and 11 to 17) have strong bioactivities indicating that the metabolites have pharmaceutical development potentiality.

Basidiosins A and B: Cyclopentapeptides from the entomophthoralean fungus Basidiobolus meristosporus.

Two new cyclopentapeptides, basidiosins A and B (1 and 2) were isolated from the mycelia extracts of entomophthoralean fungus Basidiobolus meristosporus RCEF 4516. The structures were determined based on spectroscopic methods, and the absolute config urations were assigned by Marfey's method on their acid hydrolyzates. Compounds 1 and 2 were identified as cyclo(L-Thr-L-Leu- L-Ile-D-Tyr-D-Thr) and cyclo(L-Thr-L-Leu-L-Val-D-Val-D-Ser), respectively. They were evaluated for the biological activities including antibacterial, antifungal and antioxidative activities. Furthermore, the biosynthetic pathway of 1 was proposed by bioinformatic analysis. This is the first study on the isolation of natural products from Basidiobolus fungus.

Metabolomic variation in wild and cultured cordyceps and mycelia of Isaria cicadae.

Isaria cicadae is one of the fungi used in traditional Chinese medicine with the longest tradition. It is used not only as a herbal medicine but also as a health food in Asia, together with cultured cordyceps and mycelia of the fungus used as substitute. However, the differences in their metabolite are unknown. Using a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based metabolomic method, we found that the fungus varies in its metabolism during growth on wild insects, artificially raised insects and artificial medium. There were 109 discriminatory metabolites detected in the samples by orthogonal projection to latent structure discriminant analysis and one-way ANOVA. High level of nonribosomal peptides (NRPs) only existed in the insect portions of the wild cordyceps (WI) and cultured cordyceps (CI), revealing that immunostimulation of the host insects enhanced the synthesis of NRPs in the fungus. The finding of a significantly higher level of sphingolipids in both the insect portions (WI, CI) and the coremia of the wild cordyceps (WC) and cultured cordyceps (CC) but not in cultured mycelia (CM) of I. cicadae implies that the immunostimulation of the live insects can induce the fungus to produce more sphingolipids, and this enhanced ability is probably heritable. Apart from NRPs and sphingolipids, the insect portions also contained higher levels of bioactive compounds such as lateritin, anisomycin, streptimidone and ustiloxins. In contrast, the coremium groups (WC, CC) and CM contained 10-fold less NRP but much higher levels of sanative metabolites such as tocotrienol, 3'-deoxy-hanasanagin, γ-aminobutyric acid and phospholipids than the insect portions. The significantly higher content of antioxidants in WC, CC and CM than in WI and CI suggests that environmental oxygen has a significant effect on the metabolites. The temperature stress which the wild cordyceps encounters during growth is responsible for the relatively high content of trehalose. These findings indicate that the immunity of the host insect and growth environment have a strong impact on the metabolomic variation in Isaria cicadae. The variation in metabolites suggests differential utilization value for the insect portions, coremia and mycelia of the fungus.

Metabolic responses of Beauveria bassiana to hydrogen peroxide-induced oxidative stress using an LC-MS-based metabolomics approach.

The entomopathogenic fungus, Beauveria bassiana, is commonly used as a biological agent for pest control. Environmental and biological factors expose the fungus to oxidative stress; as a result, B. bassiana has adopted a number of anti-oxidant mechanisms. In this study, we investigated metabolites of B. bassiana that are formed in response to oxidative stress from hydrogen peroxide (H2O2) by using a liquid chromatography mass spectrometry (LC-MS) approach. Partial least-squares discriminant analysis (PLS-DA) revealed differences between the control and the H2O2-treated groups. Hierarchical cluster analysis (HCA) showed 18 up-regulated metabolites and 25 down-regulated metabolites in the H2O2-treated fungus. Pathway analysis indicated that B. bassiana may be able to alleviate oxidative stress by enhancing lipid catabolism and glycometabolism, thus decreasing membrane polarity and preventing polar H2O2 or ROS from permeating into fungal cells and protecting cells against oxidative injury. Meanwhile, most of the unsaturated fatty acids that are derived from glycerophospholipids hydrolysis can convert into oxylipins through autoxidation, which can prevent the reactive oxygen of H2O2 from attacking important macromolecules of the fungus. Results showed also that H2O2 treatment can enhance mycotoxins production which implies that oxidative stress may be able to increase the virulence of the fungus. In comparison to the control group, citric acid and UDP-N-acetylglucosamine were down-regulated, which suggested that metabolic flux was occurring to the TCA cycle and enhancing carbohydrate metabolism. The findings from this study will contribute to the understanding of how the molecular mechanisms of fungus respond to environmental and biological stress factors as well as how the manipulation of such metabolisms may lead to selection of more effective fungal strains for pest control.

Differential metabolic responses of Beauveria bassiana cultured in pupae extracts, root exudates and its interactions with insect and plant.

Beauveria bassiana is a kind of world-wide entomopathogenic fungus and can also colonize plant rhizosphere. Previous researches showed differential expression of genes when entomopathogenic fungi are cultured in insect or plant materials. However, so far there is no report on metabolic alterations of B. bassiana in the environments of insect or plant. The purpose of this paper is to address this problem. Herein, we first provide the metabolomic analysis of B. bassiana cultured in insect pupae extracts (derived from Euproctis pseudoconspersa and Bombyx mori, EPP and BMP), plant root exudates (derived from asparagus and carrot, ARE and CRE), distilled water and minimal media (MM), respectively. Principal components analysis (PCA) shows that mycelia cultured in pupae extracts and root exudates are evidently separated and individually separated from MM, which indicates that fungus accommodates to insect and plant environments by different metabolic regulation mechanisms. Subsequently, orthogonal projection on latent structure-discriminant analysis (OPLS-DA) identifies differential metabolites in fungus under three environments relative to MM. Hierarchical clustering analysis (HCA) is performed to cluster compounds based on biochemical relationships, showing that sphingolipids are increased in BMP but are decreased in EPP. This observation further implies that sphingolipid metabolism may be involved in the adaptation of fungus to different hosts. In the meantime, sphingolipids are significantly decreased in root exudates but they are not decreased in distilled water, suggesting that some components of the root exudates can suppress sphingolipid to down-regulate sphingolipid metabolism. Pathway analysis finds that fatty acid metabolism is maintained at high level but non-ribosomal peptides (NRP) synthesis is unaffected in mycelia cultured in pupae extracts. In contrast, fatty acid metabolism is not changed but NRP synthesis is high in mycelia cultured in root exudates and distilled water. This indicates that fungal fatty acid metabolism is enhanced when contacting insect, but when in the absence of insect hosts NRP synthesis is increased. Ornithine, arginine and GABA are decreased in mycelia cultured in pupae extracts and root exudates but remain unchanged in distilled water, which suggests that they may be associated with fungal cross-talk with insects and plants. Trehalose and mannitol are decreased while adenine is increased in three conditions, signifying carbon shortage in cells. Together, these results unveil that B. bassiana has differential metabolic responses in pupae extracts and root exudates, and metabolic similarity in root exudates and distilled water is possibly due to the lack of insect components.

New tyrosinase inhibitors from Paecilomyces gunnii.

Through screening 50 strains of entomopathogenic fungi and rescreening of 7 strains of Paecilomyces gunnii, a methanol extract of liquid-cultivated mycelia of P. gunnii was found to have the strongest tyrosinase inhibitory activity. Preparative high-speed counter-current chromatography (HSCCC) guided by high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-high-resolution mass spectrometry (HRMS) was employed for the isolation and purification of the active components, and three new compounds with half inhibition concentration (IC50) of 0.11, 0.17, and 0.14 mM against diphenolase were obtained from the extract, respectively. Their chemical structures were identified by HRMS, one- and two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy as paecilomycones A, B, and C. Structure and activity studies showed that the tyrosinase inhibition activities are positively related to the number of hydroxyl groups on the paecilomycones.

Comparison of cytotoxic extracts from fruiting bodies, infected insects and cultured mycelia of Cordyceps formosana.

A resazurin method was employed to test and compare cytotoxicity of extracts from fruiting bodies, insects and cultured mycelia of Cordyceps formosana against Chinese hamster ovary (CHO) cells. Results showed that the cultured mycelia had much stronger cytotoxicity than that of the fruiting bodies and infected insects. This suggests that using cultured mycelia to substitute a natural Cordyceps may result in poisoning. A combined method of HPLC-PAD-HRMS and cytotoxic analysis revealed that the most toxic compound (Compound 1) was found mainly in the cultured mycelia and also a small amount in the infected insect body of the Cordyceps, but not in the fruiting body. The second toxic compound (Compound 2) was found in all structures of Cordyceps and in cultured mycelia. Different contents of the toxic compounds resulted in the different cytotoxicity of the extracts. Compound 1 and Compound 2 were prepared with preparative HPLC as yellow and orange powders, respectively. Cytotoxic tests showed that the median lethal dose (LD50) against CHO cells of Compound 1 was 18.3 ± 0.2 and 103.7 ± 5.9 µg/mL for Compound 2. Compound 1 and Compound 2 were identified as rugulosin and skyrin by HRMS, UV and NMR data. The two compounds were never previously isolated from the genera Cordyceps and Hirsutella and their cytotoxicity against CHO cells was also reported for the first time.

Metabolic effect of an exogenous gene on transgenic Beauveria bassiana using liquid chromatography-mass spectrometry-based metabolomics.

Genetic modification of Beauveria bassiana with the scorpion neurotoxin aaIT gene can distinctly increase its insecticidal activity, whereas the effect of this exogenous gene on the metabolism of B. bassiana is unknown until now. Thus, we investigate the global metabolic profiling of mycelia and conidia of transgenic and wild-type B. bassiana by liquid chromatography-mass spectrometry (LC-MS). Principal component analysis (PCA) and orthogonal projection to latent structure discriminant analysis (OPLS-DA) reveal clear discrimination of wild-type mycelia and conidia from transgenic mycelia and conidia. The decrease of glycerophospholipids, carnitine, and fatty acids and the increase of oxylipins, glyoxylate, pyruvic acid, acetylcarnitine, fumarate, ergothioneine, and trehalose in transgenic mycelia indicate the enhanced oxidative reactions. In contrast, most metabolites related to oxidative stress are not altered significantly in conidia, which implies that there will be no significant oxidative stress reaction when the aaIT gene is quiescent in cells.

Identification and production of a novel natural pigment, cordycepoid A, from Cordyceps bifusispora.

A novel yellow pigment, cordycepoid A, was isolated and identified from the entomogenous fungi Cordyceps bifusispora. Cordycepoid A exhibited no significant toxicity against Chinese hamster ovary (CHO) cells and mice, and showed high stability against food addictives, metal ions and heat. A liquid/solid double-phase cultural process for the production of the pigment was optimized as follows: 3 days aged liquid seed, 7.5 % inoculums, incubation temperature at 25 °C, 10 days of solid culture, and the last 5 days exposed to 200 Lx scattered light. The liquid seed medium and the solid culture medium were also optimized. Ethanol was selected as extracting solvent for its scale-up production. The optimal extracting conditions were determined as liquid/solid ratio at 20:1, extracting temperature at 40 °C, ultrasonic power at 400 W, and extracting time of 40 min.

[Influence of incubation time on metabolites in mycelia of Paecilomyces militaris].

OBJECTIVE: To determine the secondary metabolites production in mycelia of Paecilomyces militaris. METHODS: Mycelia were cultured in plates with sabouraud dextrose agar yeast medium at 25 degrees C for 9 days. Sampling was done every day from the second to the ninth day. The secondary metabolites in the mycelia of Paecilomyces militaris were extracted with either methanol or ethyl acetate. The extracts were blended and analyzed by liquid chromatography-mass spectrometry (LC-MS). LC-MS data were collected and analyzed by MetaboAnalyst software. RESULT: Principal component analysis indicates different secondary metabolites accumulation with incubation times. Hierarchical clustering analysis shows that the metabolic process of cationic compounds such as alkaloids, peptides and nucleosides can be divided into three stages, and that the metabolic process of anionic compounds such as organic acids and saccharides can be divided into two stages. Metabolites difference and heat map analysis show that: (1) The number of metabolites with significant increased contents was raised significantly in mycelia of Paecilomyces militaris on the second and third incubation days. The main species with increased contents were esters and their hydrolized products, destruxin B, variotin and some unidentified nitrogin contained compounds. (2) The number of metabolites with significant raised contents was decreased significantly on the fourth and fifth incubation days. The main species with increased contents were ophiocordin and destruxin A. (3) Apart from peptide antibiotics such as several beauverolides, the content increased metabolites included also several organic acids, amino acids, rhamnose, trehalose, cerebroside and riboflavine during the sixth to ninth incubation days. CONCLUSION: The secondary metabolites in mycelia of Paecilomyces militaris were related significantly to the incubation time.

[Influence of liquid or solid culture conditions on the volatile components of mycelia of Isariacateinannulata].

OBJECTIVE: To determine the volatile components of mycelia of Isaria cateinannulata cultured under different culture conditions, and to analyze the relationships between the culture conditions and volatile metabolites. METHODS: Mycelia were cultured in solid plates with SDAY medium and liquid shake flasks with SDY medium. The culture conditions were at 25 degrees C and 8 days. Volatile components in the mycelia of I. cateinannulata were extracted with simultaneous distillation extraction and analyzed by gas chromatography-mass spectrometry. RESULT: Alkenes, alkanes, heterocyclic and polycyclic aromatic hydrocarbons (PAH) were existed abundantly both in the mycelia of liquid and solid cultures, but the kinds and relative concentrations of the volatile components in mycelia of liquid and solid cultures were very different. Forty-one compounds were identified from the mycelia of solid culture and 32 compounds were identified from the mycelia of liquid culture. Esters, quinones and oximes were only found in solid cultured mycelia whereas carboxylic acids were only discovered in the mycelia of liquid culture. At the same time, mycelia of liquid culture contained much more phenols. The most abundant compounds in mycelia of liquid and solid cultures were hydrocarbons. The volatile extracts of solid cultured mycelia contained 57.6% alkenes and 9.19% alkanes. The volatile extracts of liquid cultured mycelia contained 7.85% alkenes and 22.4% alkanes. CONCLUSION: Liquid or solid culture conditions influenced the volatile components of mycelia of I. cateinannulata.

Activity-guided isolation and identification of radical scavenging components in Gao-Cha tea.

Gao-Cha is a traditional Chinese health tea made from Acer ginnala. We performed a components and radical scavenging activity analysis to identify any medicinal components in this tea. High performance thin layer chromatography (HPTLC)-1,1-Diphenyl-2-picryhydrazyl (HPTLC-DPPH) assay showed that the methanolic extract contained strong radical scavengers. Quantitative analysis revealed that the IC(50) of the extract against 1 mM DPPH was 52.7 ± 0.6 µg/mL. Bioactive-guided isolations led to procurement of 3 radical scavengers with IC(50)s of 17.5 ± 2.1, 29.3 ± 2.5, and 21.6 ± 1.7 µg/mL, respectively. Analysis of the high resolution-electric spray ionization-mass spectrometer and (1)H, (13)C, distortionless enhancement by polarization transfer at 135°, heteronuclear quantum coherence, correlating spectroscopy coupling, and heteronuclear multiple bond coherence (HMBC) data revealed that the compounds were methyl 3, 4, 5-trihydroxybenzoate (1), quercetin-3-O-α-rhamnopyranoside (2), and 2,6-bis (3,4,5-trihydroxybenzoyl)-aceritol (3). Bioactive combined components analysis revealed that, apart from compounds 1, 2, and 3, the tea possibly contained radical scavengers: ginnalin A (4) and B (5), 2″-O-Galloylquercitrin (6) and 3″-O-Galloyl-quercitrin (7). Compounds 2, 6, and 7 were isolated from Acer ginnala for the first time. The positions of the 2 galloyl moieties in compound 3 were unambiguously established by the HMBC spectrum for the first time.

[Controlled study of auricular point taping and pressing therapy for treatment of vascular dementia].

OBJECTIVE: To assess the therapeutic effect of auricular point taping and pressing therapy on vascular dementia (VD). METHODS: One hundred and eighty cases of vascular dementia were randomly divided into an auricular point taping and pressing group and a western medicine group, 90 cases in each group. The auricular point taping and pressing group was treated by auricular point taping and pressing at auricular points Shennen, Nao (brain), Shen (kidney), Zhen (pulvinal), and the western medicine group by oral administration of Nimodipine, thrice each day, 30 mg each time. They were treated for 12 weeks. The scores of mini-mental state examination (MMSE) and activities of daily living (ADL), and adverse reaction were observed. RESULTS: The scores of MMSE before treatment and 12 weeks after treatment were 18.00 +/- 3.88 and 20.83 +/- 3.74 in the auricular point taping and pressing group, and 17.80 +/- 3.82 and 20.70 +/- 3.53 in the western medicine group, respectively, with significant increases in scores of MMSE after treatment in the two groups (both P < 0.01) and with no significant difference between the two groups (P > 0.05). The scores of ADL before treatment and 12 weeks after treatment were 44.90 +/- 14.84 and 39.60 +/- 12.45 in the auricular point taping and pressing group, and 45.70 +/- 14.86 and 39.10 +/- 13.44 in the western medicine group, respectively, with significant decreases after treatment in the two groups (both P < 0.01) and with no significant difference between the two groups (P > 0.05). In the auricular point taping and pressing group, 2 cases withdrew from the test because adhesive plaster allergic reaction induced severe itch of the auricle. In the western medicine group, one case had mild dizziness and another case had diarrhea respectively. CONCLUSION: Auricular point taping and pressing and Nimodipine have similar therapeutic effect on vascular dementia, and have no obvious adverse reaction, except adhesive plaster allergic reaction.

[Anti-tumor compounds from submerger fermentation products of a Paecilomyces militaris strain].

OBJECTIVE: The aim was to identify the antitumor compounds of the fermentation broth of Paecilomyces militaris (strain RCEF0927). METHODS: Anti-tumor activity was tested with a resazurin cytotoxicity assay model with Chinese hamster ovary (CHO) cells. Bioactive compounds were analysed with combined method of High Performance Liquid Chromatography (HPLC), High Resolution Mass Spectrometer (HRMS) and antitumor assay. RESULTS: Antitumor test showed that the fermentation broth had strong cytotoxicity against CHO cells. Extraction tests showed that ethylacetate was the best solvent for the bioactive constituents extracting. HPLC-DAD- HRMS-Cytotoxicity assay revealed that the molecular formula of the active compounds in the extract were possibly C10 H13 N5 O3, C22 H22 O9, C23 H24 O10, C22 H22 O10, C15 H10 O4, C15 H10O, C36 H70 O11, C36 H68 O12, C38 H74 O11, C40 H76 O13 and C44 H82 O14. CONCLUSION: The possible structures of the bioactive compounds were deduced with bioassay, HRMS, Uv and database inquiry as cordycepin, daizein, genistein 7,4'-dihydroxyisoflavone-7-O-(4"-O-methyl)-b-D-glucopyranoside, 7, 4'-dihydroxy-6- methoxyisoflavone-7-O-( 4"-O-methyl)-b-D-glucopyranoside, 5, 7, 4'-trihydroxy-isoflavone-7-O-(4"-O-methyl)-b-D-glucopyranoside, polyoxyethylene ether oleate, dehydro products of polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxypropylene or polyoxybutene contained derivants of polyoxyethylene sorbitan fatty acid ester. Cytotoxicity of the compounds was revealed for the first time.

[Analysis and isolation of radical scavengers from metabolites of Hirsutella sp].

OBJECTIVE: During a screening for free radical scavengers from metabolites of entomogenous fungi, we fond a fermentation broth of the strain RCEF 0881 of Hirsutella sp. exhibited strong radical scavenging activity. To make clear of the constituents of the active compounds, and prepare some pure active compound for further structure identification we launched this study. METHODS: We used organic solvent for active compounds extraction. DPPH-TLC and DPPH- Microplate assay were used for activity analysis. Components analysis was carried out on a HPLC-DAD-HRMS, and bioactive compound preparation on a preparative RP-HPLC. RESULTS: Our extraction tests showed that ethylacetate was the best solvent for the bioactive constituents extracting. HPLC-DAD-HRMS-DPPH assay revealed that the molecular formular of the radical scavengers existed in the extract were possibly C7H6O4, C8H8O3 and C12H14N2O. From the chromatographic and Uv properties, and the MS fragments, and database consulting, the compounds could be deduced as dihydroxybenzoic acid, methyl-hydroxyl benzoic acid, and an alkaloid, however, the structures are still needed to be confirmed. The pick area of HPLC and MS showed that the compound C12H14N2O was the main component of the extract. It was isolated via activity directed fractionation. The activity of the prepared compound was confirmed with DPPH-TLC assay and its purity was confirmed with HPLC-DAD-HR-ESIMS. The occurance of the three active compounds in entomogenous fungi was revealed for the first time.

Free radical scavenging activity of extracts prepared from fresh leaves of selected Chinese medicinal plants.

The free radical scavenging activity of the 80% methanolic extracts from fresh leaves of 300 Chinese medical woody plants was assessed with the aid of the stable DPPH radical. Among the plants screened, 56 species had strong free radical scavenging capacities, with IC50 values lower than 0.5 mg leaves per milliliter. Analysis of the medical uses of these plants showed that most of them are employed for their effects on hemostasis, as anti-inflammatory, antimicrobial or for treatment of dysentery. These uses may be directly linked to the content in tannins and flavonoids and consequently to their free radical scavenging activities.