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Analysis of Ergot Alkaloids

B-TeZ Ergot Alkaloids Instruction Manual

BTLS327030 B-TeZ IAC Ergot alkaloids, Size 3 mL; 30/pk.
BTLS327050 B-TeZ IAC Ergot alkaloids, Size 3 mL; 50/pk.
BTLS327100 B-TeZ IAC Ergot alkaloids, Size 3 mL; 100/pk.


Extraction method with Ergosil SPE


Ergosil SPE columns (400mg/6mL, 30/pk. P/N: ERGOSIL400C)
Please ask for a quote (
or fax to: ++36 27 336375.
These columns are stock items; our preferred carrier is DPD (within the EU) or DHL (overseas).


LITERATURE (some of them are available in electronic form - please ask for it)


A rapid cleanup method for the isolation and concentration of pyrrolizidine alkaloids in comfrey root

Gray DE, Porter A, O'Neill T, Harris RK, Rottinghaus GE

J AOAC Int. 2004 Sep-Oct; 87(5):1049-57

Preparations from comfrey (Symphytum officinale and S. x uplandicum) root and leaf contain varying levels of the hepatotoxic pyrrolizidine alkaloids (PAs). Reference compounds for comfrey are not commercially available, and there is currently no rapid extraction or analytical method capable of determining low levels in raw materials or as adulterants in commercially available extracts. A solid-phase extraction (SPE) method was developed using an Ergosil cleanup column that specifically binds the PAs. With this method, powdered comfrey root was extracted by sonication and shaking with basic chloroform. The extract was applied to the cleanup column under vacuum, washed with 2 mL acetone-chloroform (8 + 2, v/v) followed by 2 mL petroleum ether to remove excess chloroform. The column was dried under vacuum, and the PAs were eluted with 2 successive 1 mL aliquots methanol. Percent recoveries of the PAs following Ergosil SPE had an overall average of 96.8%, with RSD of 3.8% over a range of 1.0 to 25.0 g extracted in 100 mL. Average precision of the method (n = 3 over 4 extraction concentrations) gave an overall RSD of 6.0% for the 5 alkaloids, with a range of 0.8% (5 g in 100 mL) to 11.2% (25 g in 100 mL). Recovery optimization testing showed that 1.0 g comfrey root extracted in 100 mL yielded the greatest recovery (% dry weight) of the PAs, with an extraction efficiency and accuracy of 94.2%, and RSD of 1.7% (n = 9). The unique properties of the Ergosil cleanup column provide rapid sample cleanup, volume reduction, and concentration of PAs from comfrey extracts, and allow the eluant to be analyzed directly by traditional chromatographic methods.



Does Mowing Height Influence Alkaloid Production in Endophytic Tall Fescue and Perennial Ryegrass?

Seppo O. Salminen1, Parwinder S. Grewal1 and Martin F. Quigley2

(1) Department of Entomology, The Ohio State University, Wooster, Ohio 44691-4096, USA

(2) Department of Horticulture and Crop Science, The Ohio State University, Columbus, Ohio 43210-1156, USA

Journal of Chemical Ecology, Volume 29, Number 6 (June 2003) pages: 1319 - 1328

The mutualistic symbiosis following infection of tall fescue, Festuca arundinacea, and perennial ryegrass, Lolium perenne, by fungal endophyte (Neotyphodium spp.) results in the production of alkaloids that are feeding deterrents or toxic to insects and livestock. If the levels of the alkaloids can be manipulated by cultural practices in the grasses that are used for home lawns and golf courses, this could alleviate the need for pesticide applications in urban environments. We evaluated the influence of mowing height on the levels of some alkaloids in a greenhouse experiment for two consecutive months. In tall fescue, levels of four of the nine alkaloids, including one presumptive alkaloid, showed increased levels with increasing the mowing height from 2.5 to 7.5 cm. The alkaloids were ergonovine, ergocryptine, perloline methyl ether, and an unidentified alkaloid designated as unknown C. In perennial ryegrass, three out of six alkaloids, perloline methyl ether, chanoclavine, and unknown A, showed similar increases. The alkaloid levels in perennial ryegrass showed more variability than those in tall fescue between the two sampling dates. It was clear in both grasses that the relative levels of the alkaloids varied with mowing height, as well as over time.



Influence of temperature on alkaloid levels and fall armyworm performance in endophytic tall fescue and perennial ryegrass

Seppo O. Salminen, Douglas S. Richmond, Sukhbir K. Grewal & Parwinder S. Grewal

Entomologia Experimentalis et Applicata, Volume 115, Issue 3, page 417 - June 2005

The symbiotic relationships between Neotyphodium endophytes (Clavicipitacea) and certain cool-season (C3) grasses result in the synthesis of several alkaloids that defend the plant against herbivory. Over a 3 month period we evaluated the effects of temperature on the expression of these alkaloids in tall fescue, Festuca arundinacea Schreb, and perennial ryegrass, Lolium perenne L. (Poaceae). Response surface regression analysis indicated that month, temperature, and their interaction had an impact on the alkaloid levels in both grasses. We aimed to identify the alkaloids most closely associated with enhanced resistance to the fall armyworm, Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae), and clarify the role of temperature in governing the expression of these alkaloids. The dry weights and survival of fall armyworms feeding on endophyte-infected tall fescue or perennial ryegrass were significantly lower than for those feeding on uninfected grass, whereas endophyte infection had no significant influence on survival. For tall fescue, a four-alkaloid model consisting of a plant alkaloid, perloline, and the fungal alkaloids ergonovine chanoclavine, and ergocryptine, explained 47% of the variation in fall armyworm dry weight, whereas a three-alkaloid model consisting of the plant alkaloid perloline methyl ether and the fungal alkaloids ergonovine and ergocryptine explained 70% of the variation in fall armyworm dry weight on perennial ryegrass. Although temperature had a significant influence on overall alkaloid expression in both grasses, the influence of temperature on individual alkaloids varied over time. The levels of those alkaloids most closely linked to armyworm performance increased linearly or curvilinearly with increasing temperature during the last 2 months of the study. We conclude that the growth temperature of grasses can influence the performance of fall armyworm, and that this effect may be mediated through a set of plant- and endophyte-related alkaloids.


Analysis of Ergot Alkaloids in Endophyte-Infected Tall Fescue by Liquid Chromatography/Electrospray Ionization Mass Spectrometry

Richard A. Shelby

Department of Plant Pathology, Auburn University, Alabama 36849

Jana Olsovska, Vladimir Havlicek, and Miroslav Flieger

Institute of Microbiology, Czech Academy of Sciences, Prague 4, Czech Republic

J. Agric. Food Chem., 45 (12), 4674 -4679, 1997.

Previous investigators have identified a number of ergot alkaloids (EAs) in tall fescue (Festuca arundinacea Schreb.) infected by the endophytic fungus, Neotyphoidium coenophialum [(Morgan-Jones & W. Gams) Glenn, Bacon & Hanlin comb. nov.]. Their results, however, may have been confounded by the presence of the related parasitic ergot fungus (Claviceps purpurea [Fr.:Fr.] Tul.), which also produces EAs. Semipreparative HPLC was used to separate fractions giving a high fluorescence response in a sample of endophyte-infected (EI) tall fescue seeds, carefully examined to eliminate the possibility of Claviceps infection. Analytical high-performance liquid chromatography (HPLC) and LC/electrospray ionization mass spectrometry (ESI-MS) were used to identify EAs after isolation. Clearly identified in the spectra were ergine, ergovaline, ergosine, ergonine, and previously undescribed EAs, didehydroergovaline and aci-ergovaline, including their epimers. Several additional EAs may have been present, but structures could not be confirmed by their spectra. Not found in the isolated fractions were ergonovine, ergotamine, or lysergylmethylcarbinolamide. The developed HPLC method was used to determine the alkaloids in plants, culms, and seeds of EI tall fescue.



Analysis of endophyte toxins: fescue and other grasses toxic to livestock

J. K. Porter

Toxicology and Mycotoxin Research Unit, Richard B. Russell Agricultural Research Center, ARS, USDA, Athens, GA 30613, USA.

Journal of Animal Science, Vol 73, Issue 3, 871-880

Research on livestock toxicoses caused by Acremonium (endophyte)-infected grasses strongly implicate the ergopeptine alkaloids with A. coenophialum-infected fescue and paxilline and the lolitrem alkaloids with A. lolii-infected perennial ryegrass as the causative agents. Isolation, identification, and detection of these toxins involves extraction with appropriate solvents, clean-up procedures, and chromatographic methods with known standards. Thin-layer, high-performance liquid and gas chromatography along with ultraviolet and mass spectrometric (i.e., electron impact, chemical ionization, tandem mass) characterizations have been reported. These methods have varying degrees of success depending on the matrix from which the alkaloids have been extracted. Ergovaline is the primary ergopeptine alkaloid isolated from cultures of A. coenophialum and also from infected fescue grass and seeds toxic to livestock. Other compounds isolated from the endophyte-infected fescue include: lysergic acid amide (ergine), the clavine class of ergot alkaloids (chanoclavine I, agroclavine, elymoclavine, penniclavine), the pyrrolizidine alkaloids (N-formylloline, N-acetylloline, N-methyloline, N-acetylnorloline), and the unique pyrrolopyrazine alkaloid peramine. The loline alkaloids and peramine have been more associated with the insect-deterrent properties of the endophyte-infected fescue than with livestock toxicoses. Also, both peramine and the ergopeptine alkaloids (ergovaline, ergotamine) have been isolated from A. lolii-infected perennial ryegrass. More recently, paxilline and lolitrem B have been detected in laboratory cultures of A. coenophialum isolated from tall fescue. The ergot alkaloids in endophyte-infected perennial ryegrass may be more related to decreased animal productivity (weight gains, reproduction problems), whereas the lolitrems cause the staggers syndrome. The detection, isolation, identification, and analyses of these compounds from Acremonium-infected grasses is presented.



Quantitative assessment of in planta distribution of metabolic activity and gene expression of an endophytic fungus

S Herd, MJ Christensen, K Saunders, DB Scott and J Schmid

Department of Microbiology and Genetics, Massey University, Palmerston North, New Zealand

Microbiology, 1997, Vol 143, 267-275

Using perennial ryegrass infected with an Acremonium transformant carrying the Escherichia coli beta-D-glucuronidase gene (gusA) (GUS system) under control of a constitutive promoter, we have developed methods for the quantitative extraction of endophyte-associated GUS activity from plant material. Fluorometric assays of these extracts allow quantitative assessment of the distribution of endophyte-associated GUS activity within single plants (tillers) with high resolution. Fluorescence microscopy with the dye Imagene Green can in addition visualize individual GUS-expressing hyphae. Since the transformant expresses the GUS gene constitutively, GUS activity can be used as an indicator of in planta endophyte metabolic activity. Using this approach we found that (i) the concentration of endophyte metabolic activity in plant tissue decreases with increasing plant size, (ii) approximately 70% of endophyte metabolic activity present in a plant is located in the leaf sheaths, (iii) basal--apical gradients and lateral (younger to older tissue) gradients of endophyte metabolic activity exist and (iv) basal--apical gradients are established early in leaf development. Our data suggest that the concentration of endophyte in each part of the plant is regulated so that a predetermined threshold of total endophyte activity per plant is not exceeded and a consistent distribution pattern is maintained.



Influence of consumption of endophyte-infested tall fescue hay on performance of heifers and lambs

J. C. Emile, S. Bony and M. Ghesquiere

Genetique et Amelioration des Plantes Fourrageres, Institut National de la Recherche Agronomique, Lusignan, France

Journal of Animal Science, 2000, Vol 78, Issue 2, 358-364

Two experiments were conducted to evaluate performance and physiological responses of heifers and lambs to Neotyphodium coenophialum-infested tall fescue hay fed under European rearing conditions. Endophyte-free (E-) or 100% endophyte-infested (E+) hay was derived from the same cultivar (cv. Clarine) so that the effect of the endophytic fungus could be clearly separated from a possible cultivar effect. In Exp. 1, starting in June 1996, 20 age- and body weight-paired Holstein dairy heifers were assigned for 97 d to one of two treatments consisting of ad libitum access to either E- or E+ hay, corresponding to 0 and .41 mg/kg ergovaline, respectively. During the experimental period, no significant difference (P>.20) in forage consumption, rectal temperature, or behavioral status of the animals was observed between the two treatments. The E+ diet induced a 10% apparent decrease in ADG and a clear reduction in prolactin (PRL) plasma concentration compared to the E- diet. When animals were all reassigned to a common endophyte-free diet, the E+ group recovered body weight and PRL to levels similar to those in animals fed E- after 7 wk. In Exp. 2, 30 Texel ram lambs were assigned to two treatments consisting of dietary E- or E+ tall fescue hay. The E- and E+ hays were harvested from the same plots as used in Exp. 1 and contained 0 and .96 mg/kg ergovaline, respectively. No effect of the endophyte was found on intake or carcass or testicle weight (P>.20) after the 95-d feeding period. The E+ treatment resulted in a slight reduction in BW at slaughter, mainly explained by a lower ruminal fill (P<.01). In E+ treated animals, prolactin concentrations dropped significantly (P<.001) from d 27. Hay assessment in both experiments showed no difference in chemical composition and IVDMD. The endophytic fungus strongly lowered the palatability of the E+ hay, although there was no effect on intake with heifers (Exp. 1) or with lambs (Exp. 2). The potential of severe heat stress, as expressed by the temperature humidity index, was not high in our experimental conditions, although they were considered rather unusually stressful for the western part of northern Europe. Yet, no economic effect on cattle was observed, in disagreement with results obtained in many previous U.S. studies.


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