Ergot alkaloids in sclerotia collected in Japan: synthetic profiles and induction of apoptosis by Clavine-type compounds.

The genus Claviceps (Clavicipitaceae) is famous for producing ergot alkaloids (EAs) in sclerotia. EAs can cause ergotism, resulting in convulsions and necrosis when ingested, making these compounds a serious concern for food safety. Agroclavine (2), a typical Clavine-type EA, is a causative agent of ergotism and is listed as a compound to be monitored by the European Food Safety Authority. Clavine-type EAs are known to cause cytotoxicity, but the mechanism has not been elucidated. We performed annexin V and PI double-staining followed by flow cytometric analysis to detect apoptosis in HepG2 and PANC-1 cells after exposure to Clavine-type EAs. Clavine-type EAs reduced cell viability and induced apoptosis in both cell lines. We then performed LC-MS analysis of EAs from 41 sclerotia samples of Claviceps collected in Japan. 24 out of 41 sclerotia extracts include peptide-type EAs (ergosine/inine: 4/4', ergotamine: 5, ergocornine/inine: 6/6', α-ergocryptine/inine: 8/8', and ergocristine/inine: 9/9') and 19 sclerotia extracts among 24 sclerotia detected peptide type EAs include Clavine-type EAs (pyroclavine: 1, agroclavine: 2, festuclavine: 3) by LC-MS. We then performed a metabolomic analysis of the EAs in the sclerotia using principal component analysis (PCA). The PCA score plots calculated for EAs suggested the existence of four groups with different EA production patterns. One of the groups was formed by the contribution of Clavine-type EAs. These results suggest that Clavine-type EAs are a family of compounds requiring attention in food safety and livestock production in Japan.

Biosynthesis, total synthesis, and biological profiles of Ergot alkaloids.

While the use of ergot alkaloids in folk medicine has been practiced for millennia, systematic investigations on their therapeutic potential began about 100 years ago. Subsequently, Albert Hofmann's discovery of lysergic acid diethylamide (LSD) and its intense psychedelic properties garnered worldwide attention and prompted further studies of this compound class. As a result, several natural ergot alkaloids were discovered and unnatural analogs were synthesized, and some were used to treat an array of maladies, including Alzheimer's and Parkinson's disease. While LSD was never commercially approved, recent clinical studies have found it can be an innovative and effective treatment option for several psychiatric disorders. Ongoing biosynthetic and total synthetic investigations aim to understand the natural origins of ergot alkaloids, help develop facile means to produce these natural products and enable their continued use as medicinal chemistry lead structures. This review recounts major developments over the past 20 years in biosynthetic, total synthetic, and pharmaceutical studies. Many ergot alkaloid biosynthetic pathways have been elucidated, with some of them subsequently applied toward "green" syntheses. New chemical methodologies have fostered a fast and efficient access to the ergoline scaffold, prompting some groups to investigate biological properties of natural product-like ergot alkaloids. Limited pharmaceutical applications have yet to completely bypass the undesirable side effects of ergotism, suggesting further studies of this drug class are likely needed and will potentially harness major therapeutic significance.

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.

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.

Toxicity of endophyte-infected tall fescue alkaloids and grass metabolites on Pratylenchus scribneri.

ABSTRACT Neotyphodium coenophialum, an endophytic fungus associated with tall fescue grass, enhances host fitness and imparts pest resistance. This symbiotum is implicated in the reduction of stresses, including plant-parasitic nematodes. To substantiate this implication, toxicological effects of root extracts, polyphenolic fraction, ergot, and loline alkaloids from endophyte-infected tall fescue were investigated using Pratylenchus scribneri, a nematode pest of tall fescue. In vitro bioassays and greenhouse studies were used as tests for effects of root fractions and compounds on motility and mortality of this lesion nematode. Greenhouse studies revealed that endophyte-infected tall fescue grasses are essentially nonhosts to P. scribneri, with root populations averaging 3 to 17 nematodes/pot, compared with 4,866 and 8,450 nematodes/pot for noninfected grasses. The in vitro assay indicated that root extracts from infected tall fescues were nematistatic. Polyphenols identified in extracts included chlorogenic acid, 3,5-dicaffeoylquinic acids, caffeic acid, and two unidentified compounds, but these were not correlated with endophyte status, qualitatively or quantitatively. Tests of several ergot alkaloids revealed that ergovaline and alpha-ergocryptine were nematicidal at 5 and 50 microg/ml, respectively, while ergocornine and ergonovine were nematistatic at most concentrations. Loline (N-formylloline), the pyrrolizidine alkaloid tested, was nematicidal (50 to 200 microg/ml). The ecological benefits of the metabolites tested here should assist in defining their role in deterring this nematode species while offering some probable mechanisms of action against plant-parasitic nematodes in general.

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.

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.

Post-genome research on the biosynthesis of ergot alkaloids.

Genome sequencing provides new opportunities and challenges for identifying genes for the biosynthesis of secondary metabolites. A putative biosynthetic gene cluster of fumigaclavine C, an ergot alkaloid of the clavine type, was identified in the genome sequence of ASPERGILLUS FUMIGATUS by a bioinformatic approach. This cluster spans 22 kb of genomic DNA and comprises at least 11 open reading frames (ORFs). Seven of them are orthologous to genes from the biosynthetic gene cluster of ergot alkaloids in CLAVICEPS PURPUREA. Experimental evidence of the identified cluster was provided by heterologous expression and biochemical characterization of two ORFs, FgaPT1 and FgaPT2, in the cluster of A. FUMIGATUS, which show remarkable similarities to dimethylallyltryptophan synthase from C. PURPUREA and function as prenyltransferases. FgaPT2 converts L-tryptophan to dimethylallyltryptophan and thereby catalyzes the first step of ergot alkaloid biosynthesis, whilst FgaPT1 catalyzes the last step of the fumigaclavine C biosynthesis, i. e., the prenylation of fumigaclavine A at C-2 position of the indole nucleus. In addition to information obtained from the gene cluster of ergot alkaloids from C. PURPUREA, the identification of the biosynthetic gene cluster of fumigaclavine C in A. FUMIGATUS opens an alternative way to study the biosynthesis of ergot alkaloids in fungi.

Electrospray[+] tandem quadrupole mass spectrometry in the elucidation of ergot alkaloids chromatographed by HPLC: screening of grass or forage samples for novel toxic compounds.

Ergot alkaloids are mycotoxins generated by grass and grain pathogens such as Claviceps, for example. Ergot alkaloid-poisoning syndromes, such as tall fescue toxicosis from endophyte-infected tall fescue grass, are important veterinary problems for cattle, horses, sheep, pigs and chickens, with consequent impact on food, meat and dairy industries. Damage to livestock is of the order of a billion dollars a year in the United States alone. HPLC with UV and fluorescence detection are the predominant means of ergot alkaloid determination, with focus on quantitation of the marker compound ergovaline, although ELISA methods are undergoing investigation. These techniques are excellent for rapid detection, but of poor specificity in defining new or poorly characterized ergot alkaloids and related compounds. This paper demonstrates the facility of using electrospray(+) mass spectrometry with multiple reaction monitoring (MRM) detection during chromatographic examination of ergot alkaloid standards of lysergic acid, lysergol, ergonovine, ergovaline, ergotamine, ergocornine, ergocryptine and ergocrystine by HPLC. Ergoline-8 position epimers could be separated on the gradient HPLC system for ergocornine, ergocrystine and ergonovine and appeared as shoulders for ergotamine and ergovaline; epimers generally showed different patterns of relative intensity for specific MRM transitions. There was reasonable correspondence between retention of standards on the 2-mm ESI(+)MS phenyl-hexyl-based reverse phase column and those on the 4-mm C18-based column. Since up to 10% of clinical cases involving toxin exposure display unidentified chromatographic peaks, 11 samples of feed components associated with such cases were studied with developed MRM methods to attempt elucidation of crucial components if possible. Ergotamine appeared in all, ergovaline appeared in five and ergocornine appeared in six; ergonovine, ergocryptine, ergocrystine and lysergol also appeared in several. In addition, molecular weights of compounds newly revealed by mass spectrometry suggested ergosine, ergostine and ergoptine in four samples, for which standards were not available. Dehydrated products of ergotamine, ergocrystine and ergocornine were discovered, along with dihydrogenated ergocrystine and ergocryptine in seven of the samples, and the issue was raised as to whether dehydration was strictly an instrument-derived artifact. Finally, five of the samples, along with fescue seed standard, evidenced one or more of 14 new ergot alkaloids ranging in size from 381 to 611 molecular weight and with key mass spectral characteristics of ergot alkaloids, specifically the pair of peaks m/z 223 and 208, corresponding to the ergoline ring system and its demethylated variant, respectively. It is anticipated that findings such as these will provide impetus to future development of analytical methodology for these heretofore relatively rare ergot alkaloid species.