New Trichothecenes Isolated from the Marine Algicolous Fungus Trichoderma brevicompactum.

Eight trichothecenes, including four new compounds 1-4 and four known entities 5-8, together with one known cyclonerane (9) were isolated from the solid-state fermentation of Trichoderma brevicompactum NTU439 isolated from the marine alga Mastophora rosea. The structures of 1-9 were determined by 1D/2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared spectroscopy) spectroscopic data. All of the compounds were evaluated for cytotoxic activity against HCT-116, PC-3, and SK-Hep-1 cancer cells by the SRB assay, and compound 8 showed promising cytotoxic activity against all three cancer cell lines with the IC50 values of 3.3 ± 0.3, 5.3 ± 0.3, and 1.8 ± 0.8 µM, respectively. Compounds 1-2, 4-6, and 7-8 potently inhibited LPS-induced NO production, and compounds 5 and 8 showed markedly inhibited gelatinolysis of MMP-9 in S1 protein-stimulated THP-1 monocytes.

Baccharin and p-coumaric acid from green propolis mitigate inflammation by modulating the production of cytokines and eicosanoids.

ETHNOPHARMACOLOGICAL RELEVANCE: Green propolis is produced by Apis mellifera honeybees using Baccharis dracunculifolia D.C. (Asteraceae) as substrate. This Southern Brazilian native plant and green propolis have been used in traditional medicine to treat gastric diseases, inflammation and liver disorders. AIM OF THE STUDY: Investigate the effects of baccharin (Bac) or p-coumaric acid (pCA) isolated from B. dracunculifolia D.C. (Asteraceae) over the inflammation induced by lipopolysaccharide (LPS) in vivo. MATERIALS AND METHODS: Inflammation was induced by LPS injection into air-pouches in mice, which were subsequently treated with Bac or pCA. Lavage fluid was collected from air pouches for the quantification of cellular influx via microscopy, and quantification of inflammatory mediators via colorimetric methods, ELISA and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: LPS-induced inflammation increased cellular influx and increased the levels of parameters related to vascular permeability and edema formation, such as nitric oxide (NO) and protein extravasation. Moreover, LPS increased the levels of cytokines and eicosanoids in the air-pouches. Importantly, both Bac and pCA suppressed the infiltration of neutrophils, production of NO and protein extravasation. Notably, the compounds promote differential regulation of cytokine and eicosanoid production. CONCLUSIONS: Our results suggest that Bac from green propolis directly affects inflammation by inhibiting the production of cytokines and eicosanoids, while pCA may exert direct, but also indirect effects on inflammation by stimulating the production of regulatory effectors such as interkeukin-10 in vivo.

Surface-enhanced Raman spectroscopywith gold nanorods modified by sodium citrate and liquid-liquid interface self-extraction for detection of deoxynivalenol in Fusarium head blight-infected wheat kernels coupled with a fully convolution network.

Surface-enhanced Raman spectroscopy (SERS) and deep learning network were adopted to develop a detection method for deoxynivalenol (DON) residues in Fusarium head blight (FHB)-infected wheat kernels. First, the liquid-liquid interface self-extraction was conducted for the rapid separation of DON in samples. Then, the gold nanorods modified with sodium citrate (Cit-AuNRs) were prepared as substrate for a gigantic enhancement of SERS signal. Results showed that the spectral characteristic peaks for DON residues of 99.5-0.5 mg/L were discernible with the relative standard deviation of 4.2%, with the limit of detection of 0.11 mg/L. Meanwhile, the fully convolutional network for the spectra of matrix input form was developed and obtained the optimal quantitative performance, with a root-mean-square error of prediction of 4.41 mg/L and coefficient of determination of prediction of 0.9827. Thus, the proposed method provides a simple, sensitive, and intelligent detection for DON in FHB-infected wheat kernels.

Exploring the dermotoxicity of the mycotoxin deoxynivalenol: combined morphologic and proteomic profiling of human epidermal cells reveals alteration of lipid biosynthesis machinery and membrane structural integrity relevant for skin barrier function.

Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most prevalent mycotoxins worldwide. Crop infestation results not only in food and feed contamination, but also in direct dermal exposure, especially during harvest and food processing. To investigate the potential dermotoxicity of DON, epidermoid squamous cell carcinoma cells A431 were compared to primary human neonatal keratinocytes (HEKn) cells via proteome/phosphoproteome profiling. In A431 cells, 10 µM DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). Mitochondrial impairment was reflected in altered metabolic competence, apparently combined with interference of the lipid biosynthesis machinery. Functional effects on the cell membrane were confirmed by live cell imaging and membrane fluidity assays (0.1-10 µM DON). Moreover, a common denominator for both A431 and HEKn cells was a significant downregulation of the squalene synthase (FDFT1). In sum, proteome alterations could be traced back to the transcription factor Klf4, a crucial regulator of skin barrier function. Overall, these results describe decisive molecular events sustaining the capability of DON to impair skin barrier function. Proteome data generated in the study are fully accessible via ProteomeXchange with the accession numbers PXD011474 and PXD013613.

Trichothecene macrolides from the endophytic fungus Paramyrothecium roridum and their cytotoxic activity.

The chemical investigation of the secondary metabolites of Paramyrothecium roridum (homotypic synonym: Myrothecium roridum), an endophytic fungus isolated from the medicinal plant Morinda officinalis, led to the isolation of twelve cytotoxic trichothecene macrolides, including two new ones, named myrothecines H and I. The structures of the new macrolides were elucidated by extensive spectroscopic measurements analyses. In addition, the cytotoxic activities of these compounds were evaluated against SF-268, NCI-H460, and HepG-2 tumor cell lines, and all isolated compounds (1-12) exhibited significant cytotoxic activity with the IC50 ranging from 0.0002-16.2 µM. Moreover, the inhibitory activity of myrothecines H and I was evidenced by inducing phosphorylation of JNK (c-Jun N-terminal protein kinase) protein and the PARP (poly ADP-ribose polymerase) cleavage, and eventually induce apoptosis of HepG-2 cells. The results indicated that myrothecines H and I could be applied as chemotherapeutic agents.

Usability of graphene oxide as a mycotoxin binder: In vitro study.

Mycotoxin management in agriculture is an essential challenge for maintaining the health of both animals and humans. Choosing the right adsorbent is still a question for many breeders and an important criterion for feed manufacturers. New adsorbents are still being sought. Graphene oxide is a promising material in the field of nanotechnology, which excels in its adsorption properties. Presented in vitro study investigates graphene oxide for the binding of mycotoxins from crushed wheat. The results show that graphene oxide has an adsorption capacity for aflatoxin 0.045 mg/g, zearalenone 0.53 mg/g and deoxynivalenol 1.69 mg/g at 37° C. In vitro simulation of crushed wheat digestion showed rapid adsorption during the gastric phase. Of the minerals, Mg, Cu and Zn were the most adsorbed. The applied dose of graphene oxide of 10 mg/g caused only a slight inhibition of the digestive enzymes α-amylase and trypsin compared to pepsin and gastric lipase. In vitro results indicated the suitability of graphene oxide in the adsorption of the aflatoxin, zearalenone and deoxynivalenol.

Antifungal trichothecene sesquiterpenes obtained from the culture broth of marine-derived Trichoderma cf. brevicompactum and their structure-activity relationship.

Two new trichothecene sesquiterpenes, trichobreols D (1) and E (2), were isolated from the culture broth of marine-derived Trichoderma cf. brevicompactum together with trichobreol A (3). The structures of 1 and 2 were assigned on the basis of their spectroscopic data. Compound 1 inhibited the growth of two yeast-like fungi, Candida albicans and Cryptococcus neoformans, with equivalent MIC values (6.3 µg/mL), while 2 gave MIC values of 12.5 and 25 µg/mL, respectively. The antifungal activities of five semisynthetic derivatives (4-8) prepared from 3 were evaluated and compared to investigate the preliminary structure-activity relationship.

Adsorption of Deoxynivalenol (DON) from Corn Steep Liquor (CSL) by the Microsphere Adsorbent SA/CMC Loaded with Calcium.

The occurrence of deoxynivalenol (DON) in animal feed is a serious issue for the livestock industry. Approaches using mycotoxin adsorbents are key to decreasing mycotoxin carryover from contaminated feed to animals. In this paper, a novel functional microsphere adsorbent comprising an alginate/carboxymethyl cellulose sodium composite loaded with calcium (SA/CMC-Ca) was prepared by an emulsification process to adsorb DON from polluted corn steep liquor (CSL) containing DON at a concentration of 3.60 µg/mL. Batch experiments were conducted under different experimental conditions: CSL volumes, reaction times, desorption times, and microsphere recyclability. Results showed that 5 g of microspheres reacted with 5 mL of DON-polluted CSL for 5 min, the microspheres can be recycled 155 times, and the maximum DON adsorption for the microspheres was 2.34 µg/mL. During recycling, microspheres were regenerated by deionized water every time; after the microspheres were cleaned, DON in the deionized water was degraded by sodium hydroxide (NaOH) at 70 °C for 1 h at pH 12. The mechanism for physical adsorption and hydrogen bonding was analyzed by scanning electron microscopy (SEM) and Fourier transform infrared spectrometry (FTIR). To the best of our knowledge, this is the first report showing that the microsphere adsorbent SA/CMC-Ca adsorbs DON. Therefore, we suggest that using microsphere absorbents would be a possible way to address DON-contaminated CSL issues in animal feed.

Iron nanoflorets on 3D-graphene-nickel: A 'Dandelion' nanostructure for selective deoxynivalenol detection.

Deoxynivalenol (DON), a cosmopolitan mycotoxin found in agricultural commodities causes serious health maladies to human and animals when accidently consumed even at a low quantity. It necessitates selective and sensitive devices to analyse DON as the conventional methods are complex and time-consuming. This study is focused on developing a selective biosensing system using iron nanoflorets graphene nickel (INFGN) as the transducer and a specific aptamer as the biorecognition element. 3D-graphene is incorporated using a low-pressure chemical vapour deposition followed by the decoration of iron nanoflorets using electrochemical deposition. INFGN enables a feasible bio-capturing due to its large surface area. The X-ray photoelectron spectroscopy analysis confirms the presence of the hydroxyl groups on the INFGN surface, which acts as the linker. Clear Fourier-transform infrared peak shifts affirm the changes with surface chemical modification and biomolecular assembly. The limit of detection attained is 2.11 pg mL-1 and displays high stability whereby it retains 30.65% of activity after 48 h. The designed INFGN demonstrates remarkable discrimination of DON against similar mycotoxins (zearalenone and ochratoxin A). Overall, the high-performance biosensor shown here is an excellent, simple and cost-effective alternative for detecting DON in food and feed samples.

Trichothecene and tremulane sesquiterpenes from a hallucinogenic mushroom Gymnopilus junonius and their cytotoxicity.

Gymnopilus junonius (Fr.) P. D. Orton (Cortinariaceae) is a hallucinogenic mushroom, a well-known poisonous mushroom that is widely known as "big laughter mushroom" because it causes excessive laughter in those who consume it. Chemical investigation of G. junonius fruiting bodies was performed, resulting in the isolation and structural identification of three sesquiterpenes (1-3), including a new trichothecene sesquiterpene (2) and a new tremulane sesquiterpene (3). Compound 1 was identified from G. junonius for the first time. The chemical structures of the new compounds were established by detailed analysis of 1D and 2D (1H-1H correlated spectroscopy [COSY], heteronuclear single quantum coherence [HSQC], and heteronuclear multiple-bond coherence [HMBC]) nuclear magnetic resonance (NMR) spectra, and high-resolution mass spectrometry (HRMS). In particular, the absolute configurations of compounds 2 and 3 were unambiguously determined by quantum chemical electronic circular dichroism (ECD) calculations. The isolated compounds (1-3) were evaluated for their cytotoxic effects on human lung and prostate cancer cell lines where trichothecene sesquiterpenes (1 and 2) showed remarkable cytotoxicity similar to that of the control drug, i.e., doxorubicin. Our findings provide experimental evidence suggesting the potential anti-cancer effects of trichothecene sesquiterpenes from a poisonous mushroom.

Cytotoxic Trichothecene Macrolides Produced by the Endophytic Myrothecium roridum.

Six new macrolides named myrothecines D-G (1-4), 16-hydroxymytoxin B (5), and 14'-dehydrovertisporin (6), including four 10,13-cyclotrichothecane derivatives, in addition to 12 known compounds (7-18), were isolated from three endophytic Myrothecium roridum, IFB-E008, IFB-E009, and IFB-E012. The isolated compounds were characterized by MS, NMR, CD, and single-crystal X-ray crystallography. The isolated macrolides exhibited an antiproliferation effect against chronic myeloid leukemia K562 and colorectal carcinoma SW1116 cell lines. Compounds 1-6 were cytotoxic, with IC50 values ranging between 56 nM and 16 µM. Since slight structural changes led to obvious activity differences, the CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) methods were then used to explore the 3D QSAR (three-dimensional quantitative structure-activity relationship) of these macrolides. The result showed that the steric, electrostatic, hydrophobic, and H-bond acceptor factors were involved in their cytotoxicity and provided an in-depth understanding of the structure-activity relationships of these metabolites.

Trichothecenes from a Soil-Derived Trichoderma brevicompactum.

Six new (1-6), together with seven known (7-13), trichothecenes were isolated from the soil-derived Trichoderma brevicompactum PSU-RSPG27. Their structures were established using spectroscopic data. The structure of 1 was confirmed by X-ray data. Trichodermin (7) exhibited the most potent activity against Plasmodium falciparum (K1 strain) with an IC50 value of 0.1 µM, while other trichothecenes (1, 8, 9, and 12) were much less active, with IC50 values in the range of 7.1-9.6 µM. Compound 7 displayed activity against noncancerous Vero cells with an IC50 value of 0.4 µM. The remaining compounds showed moderate to weak activity, with IC50 values in the range of 6.9-15.3 µM. Compounds 7 and 12 were active against human oral carcinoma (KB) cells with IC50 values of 2.4 and 3.7 µM, respectively. Additionally, compounds 7 and 12 displayed antifungal activity against Candida albicans with the respective MIC values of 1 and 2 µg/mL and were active against Cryptococcus neoformans with equal MIC values of 4 µg/mL.

Mycotoxin Incidence in Some Fish Products: QuEChERS Methodology and Liquid Chromatography Linear Ion Trap Tandem Mass Spectrometry Approach.

The inclusion of vegetal raw materials in feed for fish farming has increased the risk of mycotoxin occurrence in feed, as well as in edible tissues from fish fed with contaminated feed, due to the carry-over to muscle portions. Therefore, the objective of this study was to evaluate the occurrence of 15 mycotoxins in processed fish products, which are commonly consumed, such as smoked salmon and trout, different types of sushi, and gula substitutes. A QuEChERS method was employed to perform the mycotoxin extraction from fish samples. For mycotoxin identification and quantitation, the selected technique was the liquid chromatography-tandem mass spectrometry linear ion trap (LC-MS/MS-LIT). Smoked fish and sushi samples results were negative regarding the presence of all 15 mycotoxins studied. In contrast, small amounts of fusarenon-X and enniatin B were found in gula substitute samples.

Structure-activity relationships of trichothecenes against COLO201 cells and Cochliobolus miyabeanus: The role of 12-epoxide and macrocyclic moieties.

The novel trichothecene 12-deoxytrichodermin (3) was isolated from the fungus Trichoderma sp. 1212-03, and included with other known natural trichothecenes in a structure-activity relationship investigation against a human colon cancer cell line (COLO201) and filamentous fungus Cochliobolus miyabeanus. This revealed that the 12-epoxide functionality is critical for the cytotoxicity of simple trichothecenes trichodermin (4) and deoxynivalenol (2), while not critical for the cytotoxicity of roridin J (6) and epiisororidin E (8). In contrast, 12-epoxide is essential for the antifungal activity.

Ultrasensitive and eco-friendly immunoassays based monoclonal antibody for detection of deoxynivalenol in cereal and feed samples.

Ultrasensitive immunoassays, including an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a lateral-flow immunochromatographic assay (ICA), were developed based on a monoclonal antibody for the analysis of deoxynivalenol in food and feed samples. With 0.01 M PBS, 20% ethanol-PBS, and 60% ethanol-PBS extraction, which are environmentally safe, the 50% inhibitory concentration (IC50) and limit of detection (LOD) values were 1.83-4.68 µg/kg and 0.241-0.664 µg/kg, respectively, with recovery rates of 87.7%-137% and coefficient variation values of 3.99-9.88% (intra-assay) and 4.17-9.81% (inter-assay) for the ic-ELISA relative to the results obtained by liquid chromatography-tandem mass spectrometry (LC-MS). For the ICA strip, the visual LODs were 10-150 µg/kg, the cut-off values were 50-750 µg/kg, and the calculated LODs were 1.97-46.8 µg/kg, with different sample extraction solutions, and the recovery rates were 66.7%-127%. These methods are sensitive, simple and safe, providing an auxiliary analytical tool for screening the massive samples in markets.

Macrocyclic Trichothecene Mycotoxins from a Deadly Poisonous Mushroom, Podostroma cornu-damae.

Three new macrocyclic trichothecenes (1-3) and five known related compounds (4-8) were isolated from the MeOH extract of a plate culture of the fungus Podostroma cornu-damae, a deadly poisonous mushroom. Miophytocen D (1) is a rearranged macrocyclic type D trichothecene, featuring a bicyclo-[6.5]dodecahydrocyclopenta[ b]chromene scaffold, and the structures of new compounds (1-3) were delineated by the combination of 1D and 2D NMR spectroscopic experiments and HRESIMS, modified Mosher's esterification, and quantum chemical ECD calculations. The isolated compounds (1-8) were evaluated for cytotoxicity against four human breast cancer cell lines (Bt549, HCC70, MDA-MB-231, and MDA-MB-468). Compounds 4, 6, and 8 exhibited significant cytotoxic effects against the breast cancer cell lines, with IC50 values in the range of 0.02-80 nM, which is stronger than doxorubicin, the positive control, and a structure-activity relationship was suggested.

Optimised extraction methods for the determination of trichothecenes in rat faeces followed by liquid chromatography-tandem mass spectrometry.

The mycotoxin deoxynivalenol (DON) and some of its derivatives, such as 3­acetyl­deoxynivalenol (3AcDON), 15­acetyl­deoxynivalenol (15AcDON), deoxynivalenol­3­glucoside (DON3G) and de-epoxy deoxynivalenol (DOM-1), are commonly found in food and/or biological samples. However, literature does not present suitable methodologies for detecting and quantifying these mycotoxins at very low levels, which would be especially useful when they are present in biological samples. The main goal of the present paper was to evaluate different extraction techniques for the determination of these mycotoxins in rat faecal samples, in order to reduce the interferences present in the matrix and be able to quantify the mycotoxins at low concentration levels. Using diverse extraction methodologies such as QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) and pressurised liquid extraction (PLE), the clean-up strategy was optimised. QuEChERS extraction followed by a dispersive solid-phase extraction (dSPE) clean-up step with activated carbon was the method with the best extraction recovery results, ranging between 78% and 83% (except for DON3G). The matrix effect values were from -2% to -20% which supposed a reduction in comparison with the other tested strategies. These results enabled low quantification limits to be achieved, from 0.2 µg kg-1 to 3.4 µg kg-1. In view of the results, it was possible to quantify the natural presence of DON and DOM-1 in the tested faecal samples at low concentration levels.

Occurrence of deoxynivalenol in maize germs from North China Plain and the distribution of deoxynivalenol in the processed products of maize germs.

69 maize germ samples from North China Plain, 40 processed products of maize germs obtained in lab, 30 crude corn oils from factories and 40 refined corn oils from supermarkets in China were analyzed of deoxynivalenol (DON) by HPLC combined with ultraviolet detection and immunoaffinity column. 95.7% of maize germs were contaminated by DON. The average content was 449.0 µg/kg. The average of DON in processed products of maize germs including solvent extracted oil, cold-press oil, meal and cake was 163.7, 113.1, 1111.5 and 1175.2 µg/kg, respectively. Only 20% of crude corn oil and 12.5% of refined corn oil were contaminated by DON with the range of 67.5-340.5 µg/kg and 57.1-207.5 µg/kg, respectively. During the production of corn oil, solvent extraction oil had a larger amount of DON than pressing oil. The contamination of DON in corn oil was not serious or widespread, which indicated a low risk of health.

A peptide/maltose-binding protein fusion protein used to replace the traditional antigen for immunological detection of deoxynivalenol in food and feed.

A deoxynivalenol (DON) epitope clone (D8) was obtained by phage display technology using anti-DON monoclonal antibodies as a target molecule. Subsequently, a DON antigen mimic (D8-maltose-binding protein [MBP]) was synthesized by fusing the mimic epitope peptide with MBP. An enzyme-linked immunosorbent assay (ELISA) and urchin-like gold nanoparticle immunochromatographic assay was developed based on D8-MBP for detection of DON in maize and wheat. The half-maximal inhibitory concentration, lower detection limit, and linear range of the D8-MBP ELISA were 57.98 ±â€¯0.97, 9.83, and 11.32-286.77 ng/mL, respectively. The sensitivity of the D8-MBP ELISA was nearly 2.5 times higher than that of traditional ELISA using DON-bovine serum albumin (BSA). The detection threshold of the colloidal gold immunochromatographic assay for D8-MBP was 25 ng/mL. Thus, D8-MBP could be used to replace the traditional DON-BSA antigen for the immunological detection of DON, permitting low cost, rapid detection of DON.