Multiple regression analysis as a tool for the identification of relations between semi-quantitative LC-MS data and cytotoxicity of extracts of the fungus Fusarium avenaceum (syn. F. arthrosporioides).

The cytotoxicity of methanolic extracts from rice cultures of 53 Fusarium avenaceum strains, which had been isolated from different host organisms in Northern Europe, Canada and Australia/New Zealand, was investigated in a rat hepatoma (H4IIE-W), porcine epithelial kidney (PK-15), foetal feline lung fibroblast, dog lymphoblast (D3447), and a human hepatocarcinoma (Hep G2) cell line using the Alamar Bluetrade mark assay. All extracts were screened for known fungal metabolites using high-performance liquid chromatography with photodiode array and mass spectrometric detection, and both known and unknown metabolites were semi-quantified. Known metabolites that were determined in the cultures include acuminatopyrone, 2-amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol), antibiotic Y, aurofusarin, chlamydosporol, chlamydospordiol, enniatins, fusarin A and C, and moniliformin. Multiple regression analysis was used in order to relate fungal metabolites to the cytotoxicity of the extracts. Separate linear regression models were constructed for each cell line. Eleven different fungal metabolites were related to the cytotoxicity (P<0.05). Out of these, nine metabolites were siginificantly related to the cytotoxicity in only one of the five models, while two, namely enniatins and 2-AOD-3-ol, were significant contributors in three or four regression models, respectively. This paper describes how multiple regression analysis may be applied for the assignment of bioactivity/toxicity to the constituents of a multi-component mixture.     

Identification of cytotoxic principles from Fusarium avenaceum using bioassay-guided fractionation

The cytotoxicity of extracts from rice cultures of five Fusarium avenaceum strains against the porcine epithelial kidney cell-line PK-15 was investigated using the Alamar Blue™ assay. After the identification of known fungal metabolites, cytotoxic extracts were fractionated using semi-preparative reversed-phase HPLC and normal phase LC, and the fractions were tested for cytotoxicity. In this way, two different groups of metabolites were identified as the major cytotoxic principles of the extracts. High concentrations of enniatins, especially enniatins B and B1, inhibited the metabolic activity of PK-15 cells. Furthermore, an unidentified metabolite, produced in high amounts by a strain that produced relatively small amounts of enniatins, was also found to be cytotoxic to PK-15 cells. This study shows that enniatins, a group of cyclic depsipeptides, which have been ignored as significant contributors to the toxicity of fungal extracts, may account for most of the observed effect for F. avenaceum.     

2-Amino-14,16-dimethyloctadecan-3-ol, a new sphingosine analogue toxin in the fungal genus Fusarium

2-Amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol) was isolated from the cytotoxic rice culture extract of a strain of Fusarium avenaceum, which had previously been isolated from Norwegian grain. The structural information was obtained from LC-MS/MS, GC-MS, NMR spectroscopy and high-resolution MS data. The metabolite has a striking similarity to sphinganine, an intermediate in the biosynthesis of the sphingolipids. This similarity is a major feature of the so-called sphingosine analogue toxins; the most studied being the AAL toxins and the fumonisins. 2-AOD-3-ol was found to be cytotoxic to the rat hepatoma cell line H4IIE-W and to the porcine epithelial kidney cell line PK(15) at concentrations (EC₅₀) of 16 and 24 μM, respectively. The metabolite has been found in F. avenaceum inoculated wheat that was treated to support ideal conditions for Fusarium growth, demonstrating that the fungus has the potential to produce the metabolite under field conditions, which may occur in Northern Europe.     

Toxigenicity of enniatins from Western Australian Fusarium species to brine shrimp (Artemia franciscana)

The high prevalence (14 of 24 isolates) of enniatin-producing isolates from Western Australian Fusarium species isolated from pasture legumes associated with sheep feed refusal and rat deaths, and the high toxicity of their crude extracts to brine shrimp (Artemia franciscana) from a previous study warranted further investigation of this class of mycotoxin. Crude extracts from Fusarium acuminatum, Fusarium avenaceum, Fusarium tricinctum and Fusarium sambucinum, along with enniatins A, A1, B and B1 purified from a Western Australian strain of F. acuminatum using semi-preparative HPLC, were bioassayed using brine shrimp. All Fusarium isolates produced both enniatins B and B1, except for F. tricinctum WAC 8019, and 11 of the 17 isolates produced enniatin A1. Overall, all of the F. avenaceum isolates produced high amounts of enniatins, in particular enniatin B. One isolate of F. acuminatum (WAC 5715) and of F. tricinctum (WAC 11486) also produced high amounts of both enniatins B and B1. Only F. acuminatum WAC 5715 produced enniatin A among the tested isolates. All four purified enniatins A, A1, B, B1, individually and in combination, caused brine shrimp toxicity after 6 h of exposure, implicating that this emerging class of mycotoxin as a cause of the acute toxicity to brine shrimp observed. The mixture of all four enniatins was the most toxic to brine shrimp compared to purified individual enniatins, where the relative toxicity order was B > B1 > A1 > A. Enniatin B was the individual most toxic enniatin with some bioactivity at 5 μg/mL and almost 100% brine shrimp death at 50 μg/mL after 24 h of exposure. This study is the first report to confirm the acute toxicity of enniatins A, A1, B and B1 to brine shrimp, and also highlights the need for further investigation of the potential toxicity of these cyclic hexadepsipeptides to animals and humans.     

Goadsporin, a chemical substance which promotes secondary metabolism and morphogenesis in streptomycetes. I. Purification and characterization

Streptomycetes, which belong to the Gram-positive bacteria, produce secondary metabolites and sporulate. The timing of starting the secondary metabolite production and the sporulation depends on environmental conditions such as nitrogen and carbon sources. In order to obtain a tool for understanding the regulation mechanism, we carried out screening for chemical substances that induce secondary metabolism and sporulation in streptomycetes and found an active substance from the culture broth of Streptomyces sp. TP-A0584. This substance designated goadsporin promoted the formation of red pigment and sporulation at a concentration of 1 microM in Streptomyces lividans TK23 which does not produce the pigment under normal growth conditions. Goadsporin is an oligopeptide consisting of 19 amino acids with the molecular formula C72H97N19020S2. Sporulation and/or secondary metabolite production was induced in 36 streptomycetes strains among 42 strains tested. These results suggest that goadsporin acts on a common regulation pathway for sporulation and secondary metabolism in streptomycetes and can be a powerful tool to analyze the regulation mechanism.     

Feedborne Mycotoxins Beauvericin and Enniatins and Livestock Animals

Mycotoxins are secondary metabolites produced by several species of fungi, including the Fusarium, Aspergillus, and Penicillium species. Currently, more than 300 structurally diverse mycotoxins are known, including a group called minor mycotoxins, namely enniatins, beauvericin, and fusaproliferin. Beauvericin and enniatins possess a variety of biological activities. Their antimicrobial, antibiotic, or ionoforic activities have been proven and according to various bioassays, they are believed to be toxic. They are mainly found in cereal grains and their products, but they have also been detected in forage feedstuff. Mycotoxins in feedstuffs of livestock animals are of dual concern. First one relates to the safety of animal-derived food. Based on the available data, the carry-over of minor mycotoxins from feed to edible animal tissues is possible. The second concern relates to detrimental effects of mycotoxins on animal health and performance. This review aims to summarize current knowledge on the relation of minor mycotoxins to livestock animals.     

Mycotoxin Production in Liquid Culture and on Plants Infected with Alternaria spp. Isolated from Rocket and Cabbage

Fungi belonging to the genus Alternaria are common pathogens of fruit and vegetables with some species able to produce secondary metabolites dangerous to human health. Twenty-eight Alternaria isolates from rocket and cabbage were investigated for their mycotoxin production. Five different Alternaria toxins were extracted from synthetic liquid media and from plant material (cabbage, cultivated rocket, cauliflower). A modified Czapek-Dox medium was used for the in vitro assay. Under these conditions, more than 80% of the isolates showed the ability to produce at least one mycotoxin, generally with higher levels for tenuazonic acid. However, the same isolates analyzed in vivo seemed to lose their ability to produce tenuazonic acid. For the other mycotoxins; alternariol, alternariol monomethyl ether, altenuene and tentoxin a good correlation between in vitro and in vivo production was observed. In vitro assay is a useful tool to predict the possible mycotoxin contamination under field and greenhouse conditions.     

Anabaenopeptins from Cyanobacteria in Freshwater Bodies of Greece

Cyanobacteria are photosynthetic microorganisms that are able to produce a large number of secondary metabolites. In freshwaters, under favorable conditions, they can rapidly multiply, forming blooms, and can release their toxic/bioactive metabolites in water. Among them, anabaenopeptins (APs) are a less studied class of cyclic bioactive cyanopeptides. The occurrence and structural variety of APs in cyanobacterial blooms and cultured strains from Greek freshwaters were investigated. Cyanobacterial extracts were analyzed with LC–qTRAP MS/MS using information-dependent acquisition in enhanced ion product mode in order to obtain the fragmentation mass spectra of APs. Thirteen APs were detected, and their possible structures were annotated based on the elucidation of fragmentation spectra, including three novel ones. APs were present in the majority of bloom samples (91%) collected from nine Greek lakes during different time periods. A large variety of APs was observed, with up to eight congeners co-occurring in the same sample. AP F (87%), Oscillamide Y (87%) and AP B (65%) were the most frequently detected congeners. Thirty cyanobacterial strain cultures were also analyzed. APs were only detected in one strain (Microcystis ichtyoblabe). The results contribute to a better understanding of APs produced by freshwater cyanobacteria and expand the range of structurally characterized APs.     

Presence of Multiple Mycotoxins and Other Fungal Metabolites in Native Grasses from a Wetland Ecosystem in Argentina Intended for Grazing Cattle

The aim of this study was to evaluate the occurrence of several fungal metabolites, including mycotoxins in natural grasses (Poaceae) intended for grazing cattle. A total number of 72 and 77 different metabolites were detected on 106 and 69 grass samples collected during 2011 and 2014, respectively. A total of 60 metabolites were found across both years. Among the few mycotoxins considered toxic for ruminants, no samples of natural grasses were contaminated with aflatoxins, ochratoxin A, ergot alkaloids, and gliotoxin, among others. However, we were able to detect important metabolites (toxic to ruminants) such as type A trichothecenes, mainly T-2 toxin and HT-2 toxin (up to 5000 µg/kg each), and zearalenone (up to 2000 µg/kg), all at very high frequencies and levels. Other fungal metabolites that were found to be prevalent were other Fusarium metabolites like beauvericin, equisetin and aurofusarin, metabolites produced by Alternaria spp., sterigmatocystin and its precursors and anthrachinone derivatives. It is important to point out that the profile of common metabolites was shared during both years of sampling, and also that the occurrence of important metabolites is not a sporadic event. Considering that this area of temperate grassland is used for grazing cattle all year long due to the richness in palatable grasses (Poaceae), the present work represents a starting point for further studies on the occurrence of multi-mycotoxins in natural grasses in order to have a complete picture of the extent of cattle exposure. Also, the present study shows that the presence of zeranol in urine of beef cattle may not be a consequence of illegal use of this banned substance, but the product of the natural occurrence of zearalenone and α-zearalenol in natural grasses intended for cattle feeding.     

Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species

Beauvericin (BEA) and enniatins (ENNs) are cyclic peptide mycotoxins produced by a wide range of fungal species, including pathogenic Fusaria. Amounts of BEA and ENNs were quantified in individual rice cultures of 58 Fusarium strains belonging to 20 species, originating from different host plant species and different geographical localities. The species identification of all strains was done on the basis of the tef-1α gene sequence. The main aim of this study was to analyze the variability of the esyn1 gene encoding the enniatin synthase, the essential enzyme of this metabolic pathway, among the BEA- and ENNs-producing genotypes. The phylogenetic analysis based on the partial sequence of the esyn1 gene clearly discriminates species producing exclusively BEA from those synthesizing mainly enniatin analogues.     

Mycotoxins,Phytoestrogens and Other Secondary Metabolites in Austrian Pastures: Occurrences,Contamination Levels and Implications of Geo-Climatic Factors

Pastures are key feed sources for dairy production and can be contaminated with several secondary metabolites from fungi and plants with toxic or endocrine-disrupting activities, which possess a risk for the health, reproduction and performance of cattle. This exploratory study aimed to determine the co-occurrences and concentrations of a wide range of mycotoxins, phytoestrogens and other secondary metabolites in grazing pastures. Representative samples of pastures were collected from 18 Austrian dairy farms (one sample per farm) between April to October 2019. After sample preparation (drying and milling) the pastures were subjected to multi-metabolite analysis using LC-MS/MS. In total, 68 metabolites were detected, including regulated zearalenone and deoxynivalenol (range: 2.16–138 and 107–505 μg/kg on a dry matter (DM) basis, respectively), modified (3-deoxynivalenol-glucoside, HT-2-glucoside) and emerging Fusarium mycotoxins (e.g., enniatins), ergot alkaloids and Alternaria metabolites along with phytoestrogens and other metabolites. Aflatoxins, fumonisins, T-2 toxin, HT-2 toxin and ochratoxins were not detected. Of the geo-climatic factors and botanical diversity investigated, the environment temperature (average of 2 pre-sampling months and the sampling month) was the most influential factor. The number of fungal metabolites linearly increased with increasing temperatures and temperatures exceeding 15 °C triggered an exponential increment in the concentrations of Fusarium and Alternaria metabolites and ergot alkaloids. In conclusion, even though the levels of regulated mycotoxins detected were below the EU guidance levels, the long-term exposure along with co-occurrence with modified and emerging mycotoxins might be an underestimated risk for grazing and forage-fed livestock. The one-year preliminary data points out a dominant effect of environmental temperature in the diversity and contamination level of fungal metabolites in pastures.     

Fusarin C acts like an estrogenic agonist and stimulates breast cancer cells in vitro

Fusarin C is a mycotoxin produced by several Fusarium species and has been associated with esophageal cancer due to its carcinogenic effects. Here, we report that fusarin C stimulates growth of the breast cancer cell line MCF-7. This suggests that fusarin C can act as an estrogenic agonist and should be classified as a mycoestrogen. MCF-7 cells were stimulated in the range between 0.1 and 20 μM and inhibited when the concentration exceeded 50 μM. The toxicity of fusarin C is comparable to other mycoestrogens such as zearalenone, but the chemical structure of fusarin C is very different from other known estrogen agonists. Furthermore, the toxicity of fusarin C was tested in five additional human cell lines Caco 2, U266, PC3, MDA-MB-231 and MCF-10a which were all inhibited when the concentration of fusarin C exceeded 10 μM. To the best of our knowledge this is the first report on the mycoestrogenic properties of fusarin C.     

Toxigenic Potential of Aspergillus Species Occurring on Maize Kernels from Two Agro-Ecological Zones in Kenya

Two agro-ecological zones in Kenya were selected to compare the distribution in maize of Aspergillus spp. and their toxigenicity. These were Nandi County, which is the main maize growing region in the country but where no human aflatoxicoses have been reported, and Makueni County where most of the aflatoxicosis cases have occurred. Two hundred and fifty-five households were sampled in Nandi and 258 in Makueni, and Aspergillus was isolated from maize. Aspergillus flavus and A. parasiticus isolates were tested for the presence of aflD and aflQ genes. Positive strains were induced to produce aflatoxins on yeast extract sucrose and quantified using liquid chromatography-tandem mass spectrometry (LCMSMS). Aspergillus flavus was the most common contaminant, and the incidence of occurrence in Nandi and Makueni was not significantly different (82.33% and 73.26%, respectively). Toxigenic strains were more prevalent than non-toxigenic strains. All the toxigenic strains from Makueni were of the S-type while those from Nandi belonged to the L-type. Quantitative differences in aflatoxin production in vitro between isolates and between strains were detected with S strains producing relatively larger amounts of total aflatoxins, B toxins and lower values for G toxins. This was in accord with the frequent aflatoxicosis outbreaks in Makueni. However some L strains produced considerable amounts of B toxins. Given the widespread distribution of toxigenic strains in both regions, the risk of aflatoxin poisoning is high when favorable conditions for toxin production occur.     

TLC and HPTLC Fingerprints of Various Secondary Metabolites in the Stem of the Traditional Medicinal Climber,Solena amplexicaulis

Aim of this study was to develop a TLC and a HPTLC fingerprint profiles for various secondary metabolites of methanol extracts of the stem of the traditional medicinal climber, Solena amplexicaulis. These studies were carried out as per the methods of Harborne and Wagner et al. The profiles of various individual secondary metabolites were made and developed for authentication. The methanol extract of the stem showed the presence of 6 alkaloids, 6 flavonoids, 2 glycosides, 9 saponins and 3 terpenoids. Owing to the presence of rich variety of secondary metabolites, the stem extract of S. amplexicaulis is expected to exhibit therapeutic properties.     

The Electrospray Ionization - Mass Spectra of Erythromycin A Obtained from a Marine Streptomyces sp. Mutant

In our ongoing search for production improvements of bioactive secondary metabolites from marine Streptomyces through the induction of mutations using UV light, out of 145 isolates, mutant 10/14 was able to produce potent antibacterial metabolites other than the parent strain as established by chromatographic analysis. Up-scaling fermentation of mutant 10/14, followed by working up and isolation delivered five metabolites, phenazine, 1-acetyl-β -carboline, perlolyrin and erythromycin A, along with an oily substance. The latter two compounds were responsible for the antibacterial activity of the strain. In this article, we discuss with the mutation of the marine Streptomyces sp. AH2, bioactivity evaluation, fermentation and isolation of the microbial metabolites. Moreover, we study to first time in detail the 1D and 2D NMR and ESI MS data including ESI MS² and MS³ patterns combined with HRESI MS of erythromycin A.     

Diversity of Pea-Associated F. proliferatum and F. verticillioides Populations Revealed by FUM1 Sequence Analysis and Fumonisin Biosynthesis

Fusarium proliferatum and F. verticillioides are considered as minor pathogens of pea (Pisum sativum L.). Both species can survive in seed material without visible disease symptoms, but still contaminating it with fumonisins. Two populations of pea-derived F. proliferatum and F. verticillioides strains were subjected to FUM1 sequence divergence analysis, forming a distinct group when compared to the collection strains originating from different host species. Furthermore, the mycotoxigenic abilities of those strains were evaluated on the basis of in planta and in vitro fumonisin biosynthesis. No differences were observed in fumonisin B (FB) levels measured in pea seeds (maximum level reached 1.5 μg g⁻¹); however, in rice cultures, the majority of F. proliferatum genotypes produced higher amounts of FB₁–FB₃ than F. verticillioides strains.     

Antimicrobial Activity and Phytochemical Constituents of Leaf Extracts of Cassia auriculata

Plants produce a wide variety of phytochemical constituents, which are secondary metabolites and are used either directly or indirectly in the pharmaceutical industry. ‘For centuries, man has effectively used various components of plants or their extracts for the treatment of many diseases, including bacterial infections. In the present study methanol, chloroform and aqueous extracts of Cassia auriculata leaf were subjected for antimicrobial activity by well-diffusion method against six bacterial strains namely Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis. The results revealed that the methanol and chloroform extracts exhibited strong inhibitory activity against all the tested organisms (zone of inhibition of 12-20 mm), except Pseudomonas aeruginosa (zone of inhibition 10 mm or nil). The aqueous extracts showed moderate activity by ‘Zone of inhibition ≤12 or nil). The extracts were screened for their phytochemical constituents by standard protocols’ and were shown to contain carbohydrates, proteins, alkaloids, flavonoids, steroids, saponins and tannins. The antibacterial activity of these extracts is possibly linked to the presence of flavonoids, steroid, saponins and/or tannins. Further studies are needed to determine the precise active principles from Cassia auriculata.     

Volatile compounds in the stem bark of Sclerocarya birrea (Anacardiaceae) possess antimicrobial activity against drug-resistant strains of Helicobacter pylori

The aim of this study was to isolate and identify phytochemicals with anti-Helicobacter pylori activity from the stem bark of Sclerocarya birrea. The plant crude extract was fractionated by silica gel column and thin layer chromatography techniques, initially with ethyl acetate (EA) and subsequently with a combination of ethyl acetate/methanol/water (EMW). Further fractionation and identification of the phytoconstituents was achieved by gas chromatography and mass spectrometry (GC/MS) analysis. The antimicrobial activity of the fractions and compounds was evaluated against five metronidazole- and clarithromycin-resistant strains of H. pylori as well as a reference strain ATCC 43526 using the microbroth dilution technique. Amoxicillin was included in the experiments as a positive control antibiotic. Of the 18 fractions collected, 16 demonstrated anti-H. pylori activity with 50% minimum inhibitory concentration (MIC₅₀) values ranging from 310 μg/mL to 2500 μg/mL. Two of the fractions (EMW fraction 6 and EA fraction 1) revealed the presence of 5 and 24 compounds, respectively, representing 40.5% and 86.57% of the total composition. Most of the compounds were essential oils, with terpinen-4-ol being the most abundant agent (35.83%), followed by pyrrolidine (32.15%), aromadendrene (13.63%) and α-gurjunene (8.77%). MIC₅₀ ranges for amoxicillin, terpinen-4-ol and pyrrolidine were 0.0003-0.06 μg/mL, 0.004-0.06 μg/mL and 0.005-6.3 μg/mL, respectively. The inhibitory activities of terpinen-4-ol and pyrrolidine were similar to amoxicillin (P > 0.05). Most of these compounds are being reported in this plant for the first time and may represent new sources of therapeutically useful compounds against H. pylori.