Qualitative and Quantitative Assay of Trichothecin: a Mycotoxin Produced by Trichothecium roseum

A method for quantitative determination of trichothecin in crude culture filtrates was presented. The method utilized an agar diffusion bioassay against Candida albicans, a colorimetric test involving a halochromatic reaction with sulfuric acid, and subsequent formation of blue color with methanol, and thin-layer chromatography of trichothecin and its dinitrophenylhydrazine derivative. A positive result in all three systems confirmed the presence of trichothecin. Quantitative results were generally in close agreement.     

Cancer preventive potential of trichothecenes from Trichothecium roseum

Bioassay-guided separation of extracts from the culture broth and mycelium of the fungus Trichothecium roseum, aiming at the discovery for cancer preventive agents, resulted in the isolation of three new trichothecene sesquiterpenes, trichothecinols A-C (1-3) together with three known analogues, trichothecin (4), trichodermol (5) and trichothecolone (6). Compounds 1-6 exhibited remarkably potent inhibition against Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Further compound 1 strongly inhibited TPA-induced tumor promotion on mouse skin initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in two-stage carcinogenesis tests. These results suggest that compound 1 might be a valuable lead for further evaluation as a cancer preventive agent. In addition to their cancer preventive activity, compound 2 was found to show modest antifungal activity against Crypotcoccus albidus and Saccharomyces cerevisiae.     

Use of a polarographic method for determining trichothecin]

The polarographic behaviour of trichothecin was studied. It was shown that the antibiotic could be detected in solutions at concentrations of 7.10(-7) moles with the help of the polarographic method. Conditions for the polarographic determination of trichothecin in fermentation broth were developed. The error was not more than 3 per cent. The reliability of the results was shown by statistical treatment of data performed in accordance with the requirement of the USSR State Pharmacopeia, X ed., prescribing that the precision of the assay is such that the fiducial limits at p = 95 per cent deviate from the average value by not more than 5 per cent. Comparison of the results of trichothecin determination in the fermentation broth with the polarographic and biological methods showed no significant difference. Therefore, the polarographic method may be recommended for trichothecin determination in the fermentation broth.     

Morphogenetic action of trichothecin on Trichothecium roseum]

Trichothecin and some conditions of cultivation, especially high concentrations of carbon favour differentiation of the submerged mycelium of Trichothecium roseum, i.e. formation of submerged conidia, bud forming cells, chlamidospores, chains of barrel-shaped cells capable of germination and development of new generations of the submerged mycelium. Biosynthesis of trichothecin is connected with growth of these generations of the mycelium which are characterized by a high dehydrogenase activity. Synthesis of fibrinolytic enzymes is also possible in the period of growth of a weakly differentiated mycelium.     

Trichothecenes from the fungus Acremonium crotocinigenum BCC 20012

Two new trichothecenes (1 and 2) and a new chloroderivative of a trichothecene analogue (3) together with four known trichothecenes, crotocin, trichothecin, 8-deoxytrichothecinol B, and a trichothecene analogue, were isolated from the fungus Acremonium crotocinigenum BCC 20012. The structures of these compounds were elucidated by extensive spectroscopic analysis. Among the tested metabolites, trichothecin itself showed strongest antimalarial activity against Plasmodium falciparum K1, and cytotoxic activity against Vero cell lines with IC₅₀ values of 0.05 and 0.13 μg/mL, respectively.     

Trichothecene inactivation by proteolytic enzymes from Trichothecium roseum Lk ex Fr]

Trichothecium roseum Lk ex Fr produces simultaneously trichothecin and proteolytic enzymes possessing fibrinolytic, thrombolytic and esterase activity. In addition to the function of splitting and consuming the substrate, the proteolytic enzymes of T. roseum are probably able to hydrolize the other bond in the molecule of trichothecin, which results in partial inactivation of the antibiotic in both the fungus culture and the model experiment on trichothecin contact with the preparations of the proteolytic enzymes. Presence of proteases of T. roseum may be considered as a protective mechanism for detoxication of the metabolites toxic for the organism.     

Highly oxygenated sesquiterpenes from the liverwort Trocholejeunea sandvicensis

Four pinguisane type sesquiterpenes were isolated from the liverwort Trocholejeunea scandvicensis, together with three aromatic compounds. The structures of the cited compounds were established on the basis of spectroscopic means. The first two compounds were new sesquiterpenes, while the other compounds were previously isolated from other liverworts and lichen sp., respectively. The stereochemistry for lejeuneapinguisanolide was determined by X-ray analysis, a possible biosynthetic pathway to it was postulated. The other new sesquiterpene is lejeuneapinguisenone.     

Five New Guaiane Sesquiterpenes from the Endophytic Fungus Xylaria sp. YM 311647 of Azadirachta indica

Five new guaiane sesquiterpenes, 1 – 5 , were isolated from the culture broth of the endophytic fungus Xylaria sp. YM 311647, isolated from Azadirachta indica A. Juss . The structures of these compounds were elucidated on the basis of spectroscopic analyses, and their inhibitory activities against five pathogenic fungi were evaluated. All guaiane sesquiterpenes showed moderate or weak antifungal activities in a broth microdilution assay.     

利用GC-MS技术分析金耳发酵液挥发性成分

利用GC-MS技术对金耳发酵液中挥发性风味物质进行了分析。样品成分的质谱图与数据库(Nist库and Wiley库)中已知化合物质谱图比较。该研究证明金耳发酵液中匹配指数在800以上的倍半萜类化合物约占整个挥发性成分47%。     

Sesquiterpene coumarins from the roots of Ferula assa-foetida.

From the chloroform extract of the roots of Ferula assa-foetida, two sesquiterpene coumarins designated assafoetidnol A and assafoetidnol B were isolated, in addition to six known compounds, gummosin, polyanthin, badrakemin, neveskone, samarcandin and galbanic acid. The structures of the sesquiterpenes new compounds were established by spectroscopic methods.     

Eremophilane Sesquiterpenes and Benzene Derivatives from the Endophyte Microdiplodia sp. WGHS5

Three new eremophilane sesquiterpenes phomadecalins G−I ( 1 – 3 ) and two new benzene derivatives microdiplzenes A and B ( 12 and 13 ), together with nine known eremophilane sesquiterpenes ( 4 – 11 and 14 ) were isolated from an endophytic fungus, Microdiplodia sp. WGHS5. Their structures were elucidated by the interpretation of HR-ESI-MS and NMR data; meanwhile, the absolute configurations of new compounds were determined on the base of ECD calculations. All compounds were evaluated for the antimicrobial activities and antiproliferative effect on human gastric cancer cell lines (BGC-823).     

Endophytic fungus <Emphasis Type="Italic">Trichothecium roseum</Emphasis> LZ93 antagonizing pathogenic fungi <Emphasis Type="Italic">in vitro</Emphasis> and its secondary metabolites

The endophytic fungus Trichothecium roseum LZ93 from Maytenus hookeri was found to antagonize other pathogenic fungi in vitro. To identify which compound contributed substantially to the antagonism, we fermented the strain and purified its fermentation products. Eleven compounds were obtained, including two trichothecenes, five rosenonolactones, two cardiotonic cyclodepsipeptides, and two sterols. Compound 11β-hydroxyrosenonolactone (1) was assigned according to 1D and 2D-NMR data for the first time. At the same time, the ¹H and ¹³C-NMR assignments for 6β-hydroxyrosenonolactone (2) were revised. Of all of them, only trichothecin (6) showed strong antifungal activity. Based on our observations of the antagonistic activity and the other experimental results, we suggest that the antifungal compound trichothecin was the main contributor to the antagonistic action of T. roseum LZ93.     

New tyrosinase inhibitory sesquiterpenes from Chloranthus henryi

Chemical investigation of the leaves and stems of Chloranthus henryi resulted in the isolation and characterization of two new eudesmane-type sesquiterpenes, and two new germacrane-type sesquiterpenes, together with two known compounds. The inhibitory activities against tyrosinase of all isolates were also evaluated.     

Linderolides A-F, eudesmane-type sesquiterpene lactones and linderoline, a germacrane-type sesquiterpene from the roots of Lindera strychnifolia and their inhibitory activity on NO production in RAW 264.7 cells in vitro

From the roots of Lindera strychnifolia, seven sesquiterpenes, named linderolides A-F and linderoline, were isolated together with six known compounds. The structures of linderolides A-F were elucidated to be eudesmane-type sesquiterpenes and linderoline was found to be a derivative of a germacrane-type sesquiterpene. Their structures were elucidated by means of spectroscopic evidence and that of linderolide A was confirmed by X-ray analysis. The inhibitory activity toward NO production was assayed using RAW264.7 cells with evaluating the cell growth by MTT method, linderolides C, D and F being found to be moderately active.     

Production of volatile sesquiterpenes by Fusarium sambucinum strains with different abilities to synthesize trichothecenes.

Twenty-five strains of Fusarium sambucinum grown on wheat kernels were examined for trichothecene production and the synthesis of volatile sesquiterpenes. The volatiles were purged with air and collected on Tenax traps. Adsorbed compounds were eluted from the traps and injected into a gas chromatograph coupled with a mass spectrometer. Ten strains isolated from potato tubers produced high amounts of diacetoxyscirpenol and its derivatives. These strains were characterized by the production of high amounts of diverse sesquiterpenes. In 10 cultures, 19 compounds were detected, of which 6 were predominant and composed as much as 82% of the volatile sesquiterpene fraction (e.g., beta-farnesene, beta-chamigrene, beta-bisabolene, alpha-farnesene, trichodiene, and an unidentified compound). Fifteen strains isolated from various sources that did not produce trichothecenes produced much less volatile sesquiterpenes, with less chemical diversity. No more than six compounds were present in cultures. Two of these compounds were present in the toxigenic strains isolated from potatoes (beta-farnesene and acoradiene), but four were unique to the strains not producing trichothecenes (longifolene, isocaryophyllene, delta-elemene, and an unidentified one). The pattern of volatile sesquiterpenes was characteristic and distinctive for both toxic and nontoxic strains.     

Shochu brewing characteristics and properties of a trichothecin-resistant shochu yeast mutant

An antibiotic-resistant strain of Saccharomyces cerevisiae was isolated from shochu yeast. Three mutants were used for shochu brewing and gave higher ethanol productivities than the parent. The mutants were resistant to cycloheximide, cerulenin, trichothecin and other organic compounds such as lauric acid. In the presence of 20% (v/v) ethanol, the viability of the mutants was 87–96%, but that of the parent was 77%. Zymolyase treatment for 3 h, decreased the viability of the parent by 44% but that of the mutants only by 11–32%. Thus the higher ethanol productivity of these mutants is related to their high ethanol tolerance and resistance to various organic compounds.     

Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis

The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.     

Three types of sesquiterpenes from rhizomes of Atractylodes lancea

Eight sesquiterpenes, including four guaiane-types containing an interesting epoxy unit (1-4), a rare tricyclic carbon skeleton-type (5) and three eudesmane-types (6-8), along with five known compounds, were isolated from rhizomes of Atractylodes lancea. The structures and relative configurations of 1-8 were determined by analysis of spectroscopic data, and the absolute configuration of 8 was assigned by application of the CD technique. Compounds 1, 2 and 4 were evaluated for their cytotoxic effects against P388 and A549 cells, but all were inactive. Possible biosynthetic pathways for sesquiterpenes (1-8) were discussed.     

Inhibitory activity of eudesmane sesquiterpenes from Alpinia oxyphylla on production of nitric oxide

Sixteen eudesmane-type sesquiterpenes including seven new compounds oxyphyllanene A-G (1-2 and 5-9) were isolated from the fruits of Alpinia oxyphylla. Among them, compounds 1-2 are novel trinoreudesmane sesquiterpenes, and 5 is a noreudesmane one. Their structures were established by spectroscopic analysis, including 2D-NMR techniques. Inhibitory activity of compounds 3-8 and 10-16 were tested against nitric oxide production in LPS and IFN-γ-induced RAW 264.7 murine macrophages, and their IC(50) values ranged from 9.85 to 13.95 μg/ml.