Development of a UHPLC-FLD method for the analysis of ergot alkaloids and application to different types of cereals from Luxembourg.

Ergot alkaloids are toxins produced by some species of fungi in the genus Claviceps, that may infect rye and triticale and, in a minor degree, other types of cereals. In this study, a new UHPLC-FLD method for the quantification of the six major ergot alkaloids as well as their corresponding epimers was developed. The sample preparation was done by a solid-liquid extraction with acetonitrile and clean-up via freeze-out. The method was fully validated and then applied to 39 samples (wheat, rye, triticale, and barley) harvested in Luxembourg in 2016. Samples were sieved (1.9 × 20 mm) prior to analysis in order to remove sclerotia, hosting the alkaloids. However, 23 samples still contained at least one ergot alkaloid > LOQ and concentrations of the sum of the 6 ergot alkaloids ranged from 0.3 to 2530.1 µg/kg. Interestingly, the highest concentrations were measured in wheat and not in rye or triticale, suggesting that all kinds of cereals should be included in monitoring programs. The outcome of this study allowed giving a first overview of ergot alkaloid concentrations in cereals harvested in Luxembourg, and the measured concentrations were in similar ranges than in other parts of the world (e.g., Canada, France, Germany).

Three New Ergot Alkaloids from the Fruiting Bodies of Xylaria nigripes (Kl.) Sacc.

Three new ergot alkaloids, xylanigripones A - C (1 - 3) together with three known compounds, agroclavine (4), 8,9-didehydro-10-hydroxy-6,8-dimethylergolin (5), and (6S)-agroclavine N-oxide (6) were isolated from the fungus Xylaria nigripes (Kl.) Sacc. Their structures were elucidated by comprehensive spectroscopic analyses and high-resolution mass spectrometry as well as by comparison with the literature. The absolute configuration was determined by Density Functional Theory (DFT) calculation methods. In addition, all of the compounds were evaluated for bioactivity via a cytotoxicity assay, an acetylcholinesterase inhibition assay and a cholesterol ester transfer protein inhibition assay.

Simultaneous Determination of 25 Ergot Alkaloids in Cereal Samples by Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of 25 ergot alkaloids in cereal samples. The analytes included both -ine and -inine ergot alkaloids and were extracted using an acetonitrile and ammonium carbonate solution, followed by purification with C-18 sorbent. After full separation on a C18 column, the 25 ergot alkaloids were detected by LC-MS/MS using multiple reaction monitoring (MRM) in the positive ion mode. The linear range was 0.05-5.0 µg/kg for the 25 ergot alkaloids. The mean recoveries at three spiked concentrations varied from 76.5 to 120% with RSD < 15%. This method was validated using a FAPAS proficiency test sample of ergot alkaloids in rye flour and was finally applied to analyze real samples, including rye flours, wheat flours, whole wheat flours, bread, and noodles.

Preparative separation and purification of fumigaclavine C from fermented mycelia of Aspergillus fumigatus CY018 by macroporous adsorption resin.

In this work, the separation and purification of fumigaclavine C (FC), an ergot alkaloid with strong anti-inflammatory activity from fermented mycelia of Aspergillus fumigatus was systematically evaluated. Among the eight tested resins, the non-polar resin D101 displayed the best adsorption and desorption based on of static adsorption and desorption tests. Adsorption isotherms were constructed on D101 resin and fitted well to the Freundlich model. Dynamic adsorption and desorption tests on a column packed with D101 resin have been investigated for optimization of chromatographic parameters. Under optimized conditions, the contents of FC increased from 7.32% (w/w) in the crude extract to 67.54% in the final product with a recovery yield of 90.35% (w/w) via one run. Furthermore, a lab scale-up separation was carried out, in which the FC content and recovery yield were 65.83% and 90.13%, respectively. These results demonstrated that this adsorption-desorption strategy by using D101 resin was simple and efficient, thus showing potential for large scale purification and preparation of FC in the future.

Improving field production of ergot alkaloids by application of gametocide on rye host plants.

Ergot alkaloids are widely used in the pharmaceutical industry in drug preparations for treating migraines and Parkinson's disease, inducing uterine contraction, and other purposes. Phytopathogenic fungi of the genus Claviceps (e.g. C. purpurea) comprise a major biological source of ergot alkaloids. Worldwide industrial production of these alkaloids derives almost equally from two biotechnological procedures: submerged culture of the fungus in fermenters and field parasitic production in dormant fungal organs known as sclerotia (also termed ergot). Ergot yields from field cultivation are greatly affected by weather and also can be much reduced by pollen contamination from imperfectly male-sterile rye, as only unfertilized ovaries can be infected by C. purpurea spores. Two substances with gametocidal effect - maleic hydrazide and 2-chloroethylphosphonic acid - were tested during three consecutive seasons in small field experiments for the ability to induce or amplify the male sterility of rye as well as the impacts on germination of C. purpurea spores and general vitality of rye host plants. Maleic hydrazide was proven to be a highly effective gametocide on both a fertile rye variety and a variety with imperfectly induced cytoplasmic male sterility. It showed negligible effect on germination of C. purpurea spores. Both accurate dosaging of the active gametocidal compound and timing of the application just 2-3 weeks before onset of anthesis proved crucial to achieving high ergot yield with minimum grain impurities.

Fumigaclavine I, a new alkaloid isolated from endophyte Aspergillus terreus.

The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. Fumigaclavine I (1), a new alkaloid, was obtained, along with seven known compounds, including fumigaclavine C (2), rhizoctonic acid (3), monomethylsulochrin (4), chaetominine (5), spirotryprostatin A (6), asperfumoid (7), and lumichrome (8). The structure of compound 1 was elucidated by various spectroscopic analyses (UV, MS, 1D and 2D NMR). The in vitro cytotoxicity of compound 1 was determined by MTT assay in human hepatocarcinoma cell line SMMC-7721, showing weaker cytotoxicity, compared with cisplatin, a clinically used cancer chemotherapeutic agent.

Fumigaclavines D-H, new ergot alkaloids from endophytic Aspergillus fumigatus.

Ergot alkaloids are toxins which are produced biotechnologically on an industrial scale. The chemical investigation of endophytic Aspergillus fumigatus resulted in the isolation of five new ergot alkaloids named fumigaclavines D-H (2-6), along with three known analogues, fumigaclavine C (1), festuclavine (7), and fumigaclavine A (8). Their structures were unequivocally elucidated by extensive spectroscopic analyses in association with X-ray single-crystal diffraction. Fumigaclavines D-H are interesting clavine-type ergot alkaloids featuring a reverse prenyl moiety at C-2, with 1-4, 6, and 8 bearing additional substituents, e.g., an OH or OAc group at C-9. Compounds 2, 4, and 6-8 showed a broad spectrum of antimicrobial activity against a panel of anaerobic microorganisms, of which compounds 4 and 6 were the most active against Veillonella parvula with an MIC=16 µg/mL compared to that (0.12 µg/mL) of tinidazole, co-assayed as a positive reference.

Novel solid-phase extraction for epimer-specific quantitation of ergot alkaloids in rye flour and wheat germ oil.

Ergot alkaloids and their epimer-specific determination have gained increasing importance for food safety. A solid-phase extraction and cleanup method based on sodium-neutralized strong cation exchange (Na(+)-SCX) was developed to quantitate 12 priority ergot alkaloids in rye flour and wheat germ oil by HPLC fluorescence analysis. Sample preparation is achieved by omitting acidic and alkaline conditions enabling minimized epimerization, which is necessary to determine ergot alkaloids according to their natural distribution in foods. Ergot alkaloids are eluted from SCX-column by forming ion pairs using a sodium hexanesulfonate containing solution which prevents epimerization for at least 96 h. Method validation yielded recoveries of 80-120% (rye flour) and 71-96% (wheat germ oil) with a maximum limit of quantitation (LOQ) of 2.0 µg kg(-1) per ergot alkaloid for both matrices. The applicability of the developed method was demonstrated by analyzing 16 samples from German retail markets: 9 rye flours (max 178 ± 5 µg kg(-1)) and, reported for the first time, 7 wheat germ oils (max 56.8 ± 2.7 µg kg(-1)) expressed as the sum of 12 ergot alkaloids.

Ergot alkaloid from the sea slug Pleurobranchus forskalii.

The sea slug Pleurobranchus forskalii is a carnivorous scavenger that is widely distributed in shallow subtidal areas. Very few investigations of the chemical components of this gastropod have been reported. In this study we performed a comprehensive analysis of an extract of the marine mollusc, P. forskalii, collected off Ishigaki Island, Japan. As a result, an alkaloid was isolated from the chloroform extract. Remarkably, the structure elucidation based on the spectral data revealed that it was an ergot alkaloid peptide, ergosinine. Various ergot alkaloids have previously been isolated mainly from terrestrial higher plants or fungi. This is the first report of the isolation of an ergopeptine from marine life, and thus the known geographical extent of ergot alkaloids now includes both terrestrial and aquatic organisms.

Production, detection, and purification of clavine-type ergot alkaloids.

Ergot alkaloids are indole derivatives with diverse structures and biological activities. This chapter describes the procedure from fungal cultivation to purified ergot alkaloids, as exemplified by fumigaclavine A in Penicillium commune. Furthermore, useful notes for working with purified ergot alkaloids are given.

Chemotaxis disruption in Pratylenchus scribneri by tall fescue root extracts and alkaloids.

Tall fescue (Festuca arundinacea) forms a symbiotic relationship with the clavicipitalean fungal endophyte Neotyphodium coenophialum. Endophyte-infected grass is tolerant to nematode, but the factors responsible are unknown. One objective of this work was to determine if root extracts of tall fescue effected chemoreceptor activity of Pratylenchus scribneri by using an in vitro chemoreception bioassay. Another objective was to determine if specific ergot alkaloids (ergovaline, ergotamine, a-ergocryptine, ergonovine), and loline alkaloids, all produced by the fungal endophyte, altered chemotaxis with this bioassay. Methanolic extract from roots altered chemotaxis activities in this nematode but only from roots of plants cultured 45 > or = d, which repelled nematodes. Extracts prepared from noninfected grasses were attractants. This assay indicated that the alkaloids were either repellents or attractants. N-formylloline was an attractant at concentrations of 20 microg/ml and lower, while at higher concentrations it was a repellent. Ergovaline, the major ergot alkaloid produced by the endophyte, was repellent at both high and low concentrations and caused complete death of the nematodes.

Ergot: from witchcraft to biotechnology.

The ergot diseases of grasses, caused by members of the genus Claviceps, have had a severe impact on human history and agriculture, causing devastating epidemics. However, ergot alkaloids, the toxic components of Claviceps sclerotia, have been used intensively (and misused) as pharmaceutical drugs, and efficient biotechnological processes have been developed for their in vitro production. Molecular genetics has provided detailed insight into the genetic basis of ergot alkaloid biosynthesis and opened up perspectives for the design of new alkaloids and the improvement of production strains; it has also revealed the refined infection strategy of this biotrophic pathogen, opening up the way for better control. Nevertheless, Claviceps remains an important pathogen worldwide, and a source for potential new drugs for central nervous system diseases.

Large-scale separation of clavine alkaloids from Ipomoea muricata by pH-zone-refining centrifugal partition chromatography.

Centrifugal partition chromatography in the pH-zone-refining mode was successfully applied to the separation of alkaloids, directly from a crude extract of Ipomoea muricata. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)-acetonitrile-water (4:1:5, v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and trifluoroacetic acid (10 mM) to the aqueous mobile phase as an eluter. From 4 g of crude extract, 210 mg lysergol and 182 mg chanoclavine were obtained in 97% and 79.6% purities. Total yield recovery was >95%. Isolated alkaloids were characterized on the basis of their (1)H, (13)C NMR and ESI-MS data.

Prevalence of ergot derivatives in natural ryegrass pastures: detection and pathogenicity in the horse.

In the present study, we determined the incidence and effects of season and weather on clinical manifestations of endophyte-infected ryegrass toxicity, performed chemical detection and pharmacological bioassays on ryegrass extracts, and conducted trials on: (i) effects of domperidone or metochlopramide on ovarian inactivity induced by endophyte-infected ryegrass; (ii) efficacy of buspirone or dihydrochloro phenyl piperazine (m-CPP) for preventing suppressed milk production induced by endophyte-infected ryegrass; and (iii) efficacy of domperidone to induce ovulation during winter anestrus. Mares with toxicosis had prolonged gestation, embryonic losses, dystocia, poor mammary gland development, low milk production, prolonged uterine involution, and suppressed ovarian activity. Foals had respiratory failure, abnormalities of the skin, umbilicus, bone, and muscle, failure to thrive, blindness, testicular atrophy, and decreased serum total immunoglobulin concentrations. Endophyte-infected ryegrass and the incidence of toxicosis were correlated (r=0.861, P=0.03). Ergot alkaloids were not detected in extracts of endophyte-infected ryegrass by either thin-layer chromatography or spectrophotometry, but their presence was inferred in bioassays of extracts (dose-related increases in the contractile response of rat uterus). Mares given metoclopropamide (0.6 mg/kg/d), given orally every 8h for up to 7d) ovulated earlier (4-7d vs. 15-18d, P<0.001) than those given domperidone (1.1mg/kg/d) orally for up to 18d). Although both metoclopropamide and domperidone induced milk production, the latter did not induce ovarian cyclicity in healthy mares during seasonal anestrus. Based on these findings, we inferred that endophyte-infected ryegrass is associated with ergot alkaloid intoxication in horse.

Liquid chromatography-tandem mass spectrometry characterization of ergocristam degradation products.

The UPLC method with diode array UV detection was developed for qualitative determination of ergocristine and ergocristam including degradation products. The mechanism of the ergocristam disruptive reaction was described based on MS/MS characterization of ammonolytic product, N-(d-lysergyl)-l-valinamide (A1) and two methanolytic products, methyl ester of N-(d-lysergyl)-l-valine (M2), and N-[N-(d-lysergyl)-l-valyl]-l-phenylalanyl-d-prolyl methyl ester (M1). The influence of extraction conditions on epimerization and degradation of ergocristine and ergocristam was tested and conditions for reproducible decomposition of ergocristam were found. The presented method could potentially be applied for ergot alkaloids determination in sclerotia, fermentation broth, mycelium, and possibly contaminated food products, i.e. corn, flour, bread, etc., and feeding stuffs containing ungrounded cereals.

Clavicipitaceous fungi associated with ergoline alkaloid-containing convolvulaceae.

Ergoline alkaloids are a group of physiologically active natural products occurring in taxonomically unrelated fungal and plant taxa Clavicipitaceae (Hypocreales) and Convolvulaceae (Solanales). We show in the present paper that clavicipitaceous fungi are associated with four different ergoline alkaloid-containing plant taxa of the family Convolvulaceae. These fungi are macroscopically visible on the adaxial surface when young leaf buds are opened or are detectable by molecular biological techniques in seeds. Detectability of the fungus correlates with the absence or presence of ergoline alkaloids within the respective plant organ. The fungi contain the gene (dmaW) responsible for the committed step in ergoline alkaloid biosynthesis. Sequencing of ribosomal DNA (18S rDNA and internal transcribed spacer) as well as the dmaW gene (partial) and construction of phylogenetic trees show that the fungi are clavicipitaceous, not identical but very closely related.

Ergot and its alkaloids.

This manuscript reviews the history and pharmacognosy of ergot, and describes the isolation/preparation, chemistry, pharmacodynamics, and pharmacotherapeutics of the major ergot alkaloids and their derivatives. A brief discussion of the hallucinogenic properties of lysergic acid diethylamide is also featured. An abbreviated form of the material found in this paper is presented in a 4-hour didactic format to third-professional year PharmD students as part of their study of vascular migraine headaches, Parkinson's disease, and naturally occurring hallucinogens/hallucinogen derivatives in the modular course offering Neurology/Psychiatry.

Claviceps cyperi, a new cause of severe ergotism in dairy cattle consuming maize silage and teff hay contaminated with ergotised Cyperus esculentus (nut sedge) on the Highveld of South Africa.

During December/January 1996/97 typical summer syndrome (hyperthermia and a 30% drop in milk yield) occurred in succession in two Holstein dairy herds (n=240 and n=150 milking cows, respectively) on the South African Highveld. These farms are situated in the midst of the prime maize and dairy farming areas of South Africa where this condition had never been diagnosed before. The individual components of the concentrate on both farms were negative for ergot alkaloids. Endophytic fungi and/or ergot infestation of teff and other grasses fed to the cows were then suspected of being involved, but neither endophytes nor ergot alkaloids could be implicated from these sources. By measuring the serum prolactin levels of groups of sheep (n=5) fed the first farm's total mixed ration (TMR) or its three individual fibre components for a period of 11 days, the source of the ergot alkaloids was identified. A statistically significant decrease in the level of this hormone occurred only in the group on maize silage (which constituted 28% on dry matter base of the TMR). The involvement of the maize silage was further chemically confirmed by the high levels of total ergot alkaloids, predominantly ergocryptine, found by LC-MS in the silage as well as in the TMR (115-975 ppb and 65-300 ppb, respectively). The ergot alkaloid content (mainly ergocryptine) of the maize silage on the second affected farm was 875 ppb. Withdrawal of contaminated silage resulted in gradual recovery of stock on both farms. Nut sedge (Cyperus esculentus and Cyperus rotundus of the family Cyperaceae) has a world-wide distribution and is a common weed in annual crops, and can be parasitized by Claviceps cyperi. Careful examination of the maize silage from both farms revealed that it was heavily contaminated with nut sedge and that it contained minute sclerotia, identified as those of Claviceps cyperi, originating from the latter. Nut sedge was abundant on both farms and it is believed that late seasonal rain had resulted in mature, heavily ergotised nut sedge being cut with the silage. Claviceps cyperi sclerotia, collected on the affected fields in the following autumn contained 3600-4000 ppm ergocryptine. That the dominant alkaloid produced by this particular fungus was indeed ergocryptine, was confirmed by negative ion chemical ionization MS/MS. In one further outbreak in another Holstein herd, teff hay contaminated with ergotised nut sedge and containing 1200 ppb alkaloids, was incriminated as the cause of the condition. This is the first report of bovine ergotism not associated with the Poaceae infected with Claviceps purpureum or endophytes but with the family Cyperaceae and this particular fungal phytopathogen.