Applying Molecular Networking for the Detection of Natural Sources and Analogues of the Selective Gq Protein Inhibitor FR900359.

The cyclic depsipeptide FR900359 (FR), isolated from the traditional Chinese medicine plant Ardisia crenata, is a potent Gq protein inhibitor and thus a valuable tool to study Gq-mediated signaling of G protein-coupled receptors. Two new FR analogues (3 and 4) were isolated from A. crenata together with the known analogues 1 and 2. The structures of compounds 3 and 4 were established by NMR spectroscopic data and MS-based molecular networking followed by in-depth LCMS2 analysis. The latter approach led to the annotation of further FR analogues 5-9. Comparative bioactivity tests of compounds 1-4 along with the parent molecule FR showed high-affinity binding to Gq proteins in the low nanomolar range (IC50 = 2.3-16.8 nM) for all analogues as well as equipotent inhibition of Gq signaling, which gives important SAR insights into this valuable natural product. Additionally, FR was detected from leaves of five other Ardisia species, among them the non-nodulated leaves of Ardisia lucida, implying a much broader distribution of FR than originally anticipated.

Biological characteristics, bioactive components and antineoplastic properties of sporoderm-broken spores from wild Cordyceps cicadae.

BACKGROUND: Cordyceps cicadae, an entomogenous fungus has been used as a dietary therapeutic in traditional Chinese medicine for several millennia, in the form of powders and decoction. However, wild C. cicadae is notably scarce. To date, there is still a lack of comprehensive and deep studies on the biological characteristics, chemical profiles and antineoplastic mechanisms of C. cicadae, especially its spores. AIM OF THE STUDY: This study aimed to identify wild C. cicadae using rDNA-ITS sequences. Active constituents and volatile ingredients of C. cicadae sporoderm-broken spore powders (CCBSP) were elucidated using UPLC-ESI-Q-TOF-MS and GC-MS, respectively. The underlying anti-neoplastic mechanisms of CCBSP were further investigated in A549 lung carcinoma cells. RESULTS: Molecular phylogenetic analysis of nuclear rDNA sequences indicated that wild C. cicadae belonged to Paecilomyces cicadae. Eight primary compounds from CCBSP were identified by MS fragmentation ions including nucleosides, cordycepic acid, cordycepin, beauvericin and myriocin. In total, forty-nine volatile components representing 99.56% of CCBSP were clearly identified. CCBSP exhibited antiproliferative effects on A549 cells with IC50 value of 125.54 ± 2.71 µg/ml, blocking the cell cycle in the G2/M phase. The nuclear morphology exhibited typical characteristics of apoptosis by Hoechst fluorescent stain. AnnexinV-FITC/PI staining revealed that the number of apoptotic cells increased after CCBSP treatment. Furthermore, immunofluorescence experiments indicated that CCBSP lowered the expressions of ß-catenin and N-cadherin, which was accompanied by repressed Wnt/ß-catenin signalling and activation of caspase-mediated apoptosis pathways. CONCLUSIONS: rDNA-ITS sequencing enabled molecular identification of wild C. cicadae. Importantly, these findings provide the first evidence regarding the full-scale bioactive components and antineoplastic properties of CCBSP. These data highlight the significance of C. cicadae as a potential antineoplastic agent.

Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth.

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02% trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2% to 20%, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

Mycotoxins in Plant-Based Dietary Supplements: Hidden Health Risk for Consumers.

Mycotoxin contamination of dietary supplements represents a possible risk for human health, especially in the case of products intended for people suffering from certain health conditions. The aim of this study was to assess the extent of this problem based on analyses of a wide set of herbal-based dietary supplements intended for various purposes: (i) treatment of liver diseases (milk thistle); (ii) reduction of menopause effects (red clover, flax seed, and soy); and (iii) preparations for general health support (green barley, nettle, goji berries, yucca, etc.) The analytical method including 57 mycotoxins was based on a QuEChERS-like (quick, easy, cheap, effective, rugged, safe) approach and ultrahigh performance liquid chromatography coupled with tandem mass spectrometry. The main mycotoxins determined were Fusarium trichothecenes, zearalenone and enniatins, and Alternaria mycotoxins. Co-occurrence of enniatins, HT-2/T-2 toxins, and Alternaria toxins was observed in many cases. The highest mycotoxin concentrations were found in milk thistle-based supplements (up to 37 mg/kg in the sum).

Evaluation of immunologic effect of Enniatin A and quantitative determination in feces, urine and serum on treated Wistar rats.

Study of dietary supplementation with ENN A mycotoxin during 28 days of exposure time on Wistar rats to determinate its levels in serum, urine and feces and, to evaluate the immunologic effect in peripheral blood lymphocytes (PBL) is presented. The first method for ENN A extraction, determination and detection by LC-MS/MS in serum, urine and feces samples is reported. ENN A food dose administrated was detected in serum samples and influenced lymphocyte phenotyping. Levels in serum were founded from the second week of the experiment; reaching values of 4.76 µg/ml on the fourth week, which corresponds to 3.24 µg/ml in blood. PBL as T helper (CD4(+)) were presented in greater percentages compared to control (p ≤ 0.001), while T cytotoxic (CD8(+)) decreased significantly compared to control (p ≤ 0.001). ENN A treatment significantly increased CD4(+)/CD3(+) and CD4(+)/CD8(+) ratios but significantly decreased CD8(+)/CD3(+) ratio. CD4(+)/CD8(+) ratio was 2.94:1, indicating that PBL surface antigen expression and immune status in Wistar rats treated were impaired by the ENN A mycotoxin.

Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs.

A total of 60 Chinese medicinal herbs were examined for contamination of the emerging Fusarium mycotoxins enniatins (ENNs) A, A1, B, B1 and beauvericin (BEA). The herbs under study are commonly used in China as both medicines and food. The dried samples of herbs were randomly collected from traditional Chinese medicine stores in Zhejiang province, China. Sample preparation was achieved by methanol extraction, followed by a simple membrane filtration step; no tedious clean-ups were involved. ENNs A, A1, B, B1 and BEA were analysed by the recently developed stable isotope dilution assays, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With limits of detection ranging between 0.8 and 1.2 µg kg(-1) for the analytes under study, 25% of all analysed samples were contaminated with at least one of the ENNs and BEA. BEA was the most frequently detected toxin with a 20% incidence in all samples. The percentages of ENN-positive samples were lower: each single ENN was detected in 6.7-11.7% of all samples. Considering the total amounts of the five mycotoxins in single samples, values between 2.5 and 751 µg kg(-1) were found. The mean total amount in positive samples was 126 µg kg(-1). Regarding ginger, the frequent occurrence of ENNs and BEA in dried ginger could be confirmed in samples from Germany. However, in fresh ginger root the toxins were not detectable. This is the first report on the presence of ENNs and BEA in Chinese medicinal herbs.

Ciclohexadespipeptide beauvericin degradation by different strains of Saccharomyces cerevisiae.

The interaction between the mycotoxin beauvericin (BEA) and 9 yeast strains of Saccharomyces cerevisiae named LO9, YE-2, YE5, YE-6, YE-4, A34, A17, A42 and A08 was studied. The biological degradations were carried out under aerobic conditions in the liquid medium of Potato Dextrose Broth (PDB) at 25°C for 48 h and in a food/feed system composed of corn flour at 37°C for 3 days, respectively. BEA present in fermented medium and corn flour was determined using liquid chromatography coupled to the mass spectrometry detector in tandem (LC-MS/MS) and the BEA degradation products produced during the fermentations were determined using the technique of the liquid chromatography coupled to a linear ion trap (LIT). Results showed that the S. cerevisiae strains reduced meanly the concentration of the BEA present in PDB by 86.2% and in a food system by 71.1%. All the S. cerevisiae strains used in this study showed a significant BEA reduction during the fermentation process employed.

Mycotoxins produced by Fusarium spp. associated with Fusarium head blight of wheat in Western Australia.

An isolated occurrence of Fusarium head blight (FHB) of wheat was detected in the south-west region of Western Australia during the 2003 harvest season. The molecular identity of 23 isolates of Fusarium spp. collected from this region during the FHB outbreak confirmed the associated pathogens to be F. graminearum, F. acuminatum or F. tricinctum. Moreover, the toxicity of their crude extracts from Czapek-Dox liquid broth and millet seed cultures to brine shrimp (Artemia franciscana) was associated with high mortality levels. The main mycotoxins detected were type B trichothecenes (deoxynivalenol and 3-acetyldeoxynivalenol), enniatins, chlamydosporol and zearalenone. This study is the first report on the mycotoxin profiles of Fusarium spp. associated with FHB of wheat in Western Australia. This study highlights the need for monitoring not just for the presence of the specific Fusarium spp. present in any affected grain but also for their potential mycotoxin and other toxic secondary metabolites.

Characterization and mycotoxigenic potential of Fusarium species in freshly harvested and stored sugar beet in Europe.

Based on a 2-year field trial at two locations in Lower Saxony (Germany), 395 Fusarium isolates belonging to 13 species were collected from more than 3,000 sugar beet roots that were apparently healthy at harvest. In a comparative screen, subsamples were analyzed for Fusarium infection directly after harvest and after different storage conditions. Depending on the storage duration, a different species composition was observed. F. redolens was predominant in freshly harvested beets, while F. culmorum, F. cerealis, and F. graminearum comprised 50.0% (2006) and 84.8% (2007) of the Fusarium mycoflora of sugar beets subjected to long-term pile storage. Randomly selected isolates of all species detected were tested for pathogenicity to sugar beet, but only isolates of F. graminearum and F. sambucinum caused severe root symptoms. Overall, 34 isolates of all species detected were characterized for their mycotoxin profile in rice culture to determine potentially produced toxins for future analysis of sugar beet. A total of 26 Fusarium mycotoxins were detected by liquid chromatography-tandem mass spectrometry, including trichothecenes, zearalenone, and especially high amounts of beauvericin, enniatins, and moniliformin. Further work is required to analyze the natural occurrence of these mycotoxins in sugar beet.

Pathogenicity, symptom development, and mycotoxin formation in wheat by Fusarium species frequently isolated from sugar beet.

Crop rotations with putative non-host crops such as sugar beet are often recommended to reduce Fusarium head blight (FHB) in cereals. However, recent observations have shown pathogenic, endophytic, and saprotrophic colonization of sugar beet with various Fusarium spp. Therefore, strains of seven species frequently isolated from sugar beet were tested for pathogenicity on wheat. Species-specific symptoms on heads and kernels were evaluated and the grains were analyzed for 20 mycotoxins with liquid chromatography-tandem mass spectrometry. Fusarium graminearum, F. culmorum, and F. cerealis from sugar beet caused typical FHB symptoms and mycotoxin contamination with deoxynivalenol and nivalenol, while a high incidence of black point was observed in heads inoculated with F. tricinctum or F. equiseti. Black point kernels revealed 3.4 to 14.5 times higher mycotoxin concentrations than symptomless grains, containing enniatin B1 at 38,000 µg/kg, moniliformin at 4,900 µg/kg, and 2-amino-14,16-dimethyloctadecan-3-ol at 5,500 µg/kg, as well as monoacetoxyscirpenol at 2,600 µg/kg and nivalenol at 3,800 µg/kg. Monitoring of these latter two species in the field is hampered by the lack of typical head symptoms after infection. In further experiments, the impact of sugar beet residues on FHB severity and the correlation between mycotoxin contamination of cereal lots and the amount of black point have to be evaluated.

Development of a multi-mycotoxin liquid chromatography/tandem mass spectrometry method for sweet pepper analysis.

A multi-mycotoxin method was developed for the simultaneous determination of trichothecenes (nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin, T-2 toxin), aflatoxins (aflatoxin-B(1), aflatoxin-B(2), aflatoxin-G(1) and aflatoxin-G(2)), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B(1), fumonisin-B(2) and fumonisin-B(3)), ochratoxin A, zearalenone, beauvericin and sterigmatocystin in sweet pepper. Sweet pepper was extracted with ethyl acetate/formic acid (99:1, v/v). After splitting up the extract, two-thirds of the extract was cleaned up using an aminopropyl column followed by an octadecyl column. The remaining part was cleaned up using a strong anion-exchange column. After recombination of both cleaned parts of the sample extract, the combined solvents were evaporated and the residue was dissolved in mobile phase; 20 microL was injected into the chromatographic system, so only one run was used to separate and detect the mycotoxins in positive electrospray ionization using selected reaction monitoring. The samples were analyzed with a Micromass Quattro Micro triple quadrupole mass spectrometer (Waters, Milford, MA, USA). The mobile phase consisted of variable mixtures of water and methanol, 1% acetic acid and 5 mM ammonium acetate. The limits of detection of the multi-mycotoxin method varied from 0.32 microg kg(-1) to 42.48 microg kg(-1). The multi-mycotoxin liquid chromatography/tandem mass spectrometry (LC/MS/MS) method fulfilled the method performance criteria required by the Commission Regulation (EC) No 401/2006. Sweet peppers inoculated by Fusarium species were analyzed using the developed method. Beauvericin (9-484 microg kg(-1)) and fumonisins (fumonisin-B(1) up to 4330 microg kg(-1), fumonisin-B(2) up to 4900 microg kg(-1), and fumonisin-B(3) up to 299 microg kg(-1)) were detected.

Mass spectrometric studies on the intrinsic stability of destruxin E from Metarhizium anisopliae.

Destruxins are of current interest as bioactive agents. They are cyclic hexadepsipeptides produced by fungi, the most common destruxins, A, B and E, differing in the structure of a side chain. Before they can be widely used, the potential risk of destruxins and their metabolites entering the human food chain must to be assessed; thus, knowledge of the structures of their degradation products is essential. Here we report a study aimed at identifying, by tandem mass spectrometry and accurate mass analysis, the products resulting from thermally and temporally induced degradation of destruxin E. The degradation products fell into two groups: those with relatively simple modifications of the side chain and those involving much more complex rearrangements. The structures of most of the degradation products were deduced from the MS data, with the major product being destruxin E diol: significantly, this compound had previously been reported to have only been produced as a metabolic product of enzyme action rather than as a simple degradation product as demonstrated here.