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.

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.

Occurrence of peptide and clavine ergot alkaloids in tall fescue grass.

Evidence is presented that ergot alkaloids are ubiquitous in tall fescue pastures infected with the clavicipitaceous fungal endophyte Sphacelia typhina (or Acremonium coenophialum). Ergopeptide alkaloids, predominantly ergovaline, constituted 10 to 50 percent of the total ergot alkaloid concentration, which was as high as 14 milligrams per kilogram in sheaths and 1.5 milligrams per kilogram in blades. Ergot alkaloid concentrations were substantially increased by application of large amounts (10 millimoles per liter) of potassium nitrate or ammonium chloride to infected plants in the greenhouse. The results indicate that ergot alkaloids are probably responsible for the toxicity to cattle of this common pasture and lawn grass and that ergotism-like toxicoses may be caused by clavicipitaceous fungi other than Claviceps.

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.

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.

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).

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.

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.

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.

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.

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.

A novel ergot alkaloid as a 5-HT(1A) inhibitor produced by Dicyma sp.

In the course of a search for small-molecule inhibitors of 5-hydroxytryptamine receptors we have identified a novel ergoline derivative (1) from the fungal culture of Dicyma sp. This compound has a pK(i) of 10.2 versus the 5-hydroxytryptamine(1A) receptor subtype. The structure was elucidated by extensive NMR spectroscopy and mass spectrometry.

Separation of ergot alkaloids and their epimers and determination in sclerotia by capillary electrophoresis.

Capillary electrophoresis has been shown to be a very useful analysis technique for secondary metabolites of plants. In the present study a method is described for the qualitative and quantitative determination of ergot alkaloids and their epimers. The extraction from the biological matrix yields recoveries of 50-97%, depending on the individual alkaloid. Using a mixture of 20 mM beta-cyclodextrin (CD), 8 mM gamma-CD, 2 M urea and 0.3% (w/w) poly(vinyl alcohol) to phosphate buffer at pH 2.5 the simultaneous separation of all analytes was achieved. A 37 cm (30 cm) fused-silica capillary, at a voltage of 25 kV and a temperature of 20 degrees C, was used for the analysis. Overall analysis time for the separation was 12 min. The limit of detection of the alkaloids using UV detection at 214 nm can be improved 30-fold to about 9.10(-8) M when laser-induced fluorescence detection is applied.

The obstetrical use in ancient and early modern times of Convolvulus scammonia or Scammony: another non-fungal source of ergot alkaloids?

PIP: This brief historical survey documenting the use of ergot alkaloids throughout Ancient times for obstetrical purposes (labor induction and control of uterine hemmorrhage) takes its data from works of Theophrastus (who wrote Enquiry into Plants around 300 B.C.), Pliny, Dioscorides, and others. What this history documents is another source for the ergot besides the fungus Clavicips purpurea; notations from Greek, Mexican, and English catalogers show that the family Convolvulaceae is a constant source of ergot alkaloids; this family is the home of Rivea corymbosa, the morning glory seed used by Mexican Indians for both medicinal and religious purposes. In addition, an argument made from salient quotations from historical texts indicates that Scammony may also contain ergot alkaloids, although such compounds have yet to be chemically isolated. Only 1 extant study intimates the possible existence of ergot-like activity in Scammony, a study which showed that small doses of an ether extract of Scammony resin stimulated an isolated guinea pig uterus. If Scammony does contain ergot alkaloids, it is suggested that it will be an excellent source for this material, since the plants cultivation is inexpensive and easy.^ieng

Ergot alkaloids--sources, structures and analytical methods.

Natural sources, i.e. fungal strains and species producing ergot alkaloids (EA), are surveyed together with the chemical structures of EA and a list of new natural EA discovered in the last three decades. Other topics include new efficient chromatographic methods (HPLC) for the separation and isolation of new natural EA and also immunological methods of EA detection.

Compounds isolated at the Institute of Microbiology in 1989-2001 and future trends.

A total of 307 new compounds, natural, semisynthetic or synthetic, were isolated at the Institute of microbiology during the last twelve years. Due to the development of separation (chromatographic) methods and of analytical methods used to determine the chemical structure of these compounds, i.e. NMR, MS and X-ray diffraction, many new metabolites could be described.

Compounds isolated at the Department of Biogenesis of Natural Substances, Institute of Microbiology, Czechoslovak Academy of Sciences, in 1984-1988.

A total of 41 compounds studied at the Department of Biogenesis of Natural Substances between 1984 and 1988 are characterized and tabulated. They include natural, semisynthetic and synthetic compounds.

Structural elucidation of two novel ergot alkaloid impurities in alpha-ergokryptine and bromokryptine.

Two novel natural derivatives (2 and 3) of the ergot alkaloid alpha-ergokryptine (1), as well as their synthetically brominated analogues (5 and 6) were isolated and identified by NMR and MS methods. Compounds 2 and 5 contain what appears to be a so far unknown natural amino acid building block. Complete 1H and 13C NMR assignments are given for compounds 1-6.

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.