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Analysis of herbal teas made from the leaves of comfrey (Symphytum officinale): reduction of N-oxides results in order of magnitude increases in the measurable concentration of pyrrolizidine alkaloids.

OBJECTIVES: To determine the relative quantities of two hepatotoxic pyrrolizidine alkaloids, symphytine and echimidine, in teas prepared from comfrey leaves (Symphytum officinale), and to determine the potential contribution of the N-oxide forms of these alkaloids to levels of the parent alkaloids. DESIGN: Comfrey leaves were purchased from three commercial sources and used to prepare tea in a manner consistent with the methods used by consumers. An extraction scheme was devised for extraction of the alkaloids, and a gas chromatographic method was developed to quantify the two major alkaloids, symphytine and echimidine. Recognising that the N-oxide derivatives of these alkaloids have also been identified in comfrey preparations, chemical reduction was applied to determine the total quantities of the alkaloids as free bases and as N-oxide derivatives. RESULTS: The concentration of symphytine and echimidine varied considerably between teas prepared from leaves purchased from the different vendors of plant material. Moreover, a much higher concentration of symphytine was found in the tea when steps were included to reduce N-oxides prior to analysis. The treatment of pure symphytine with hot water did not generate the N-oxide derivative de novo. CONCLUSIONS: Since the pyrrolizidine alkaloids are known to be hepatotoxic, consumption of herbal teas made from comfrey leaves may be ill-advised. The concentration of pyrrolizidine alkaloids in such teas may be underestimated substantially unless the concentration of N-oxides is taken into consideration.

Beverages↗

Correlation between polyamines and pyrrolidine alkaloids in developing tobacco callus.

Since the diamine putrescine can be metabolized into the pyrrolidine ring of tobacco alkaloids as well as into the higher polyamines, we have investigated the quantitative relationship between putrescine and these metabolites in tobacco callus cultured in vitro. We measured levels of free and conjugated putrescine and spermidine, and pyrrolidine alkaloids, as well as activities of the putrescine-biosynthetic enzymes arginine and ornithine decarboxylase. In callus grown on high (11.5 micromolar) alpha-naphthalene acetic acid, suboptimal for alkaloid biosynthesis, putrescine and spermidine conjugates were the main putrescine derivatives, while in callus grown on low (1.5 micromolar) alpha-naphthalene acetic acid, optimal for alkaloid formation, nornicotine and nicotine were the main putrescine derivatives. During callus development, a significant negative correlation was found between levels of perchloric acid-soluble putrescine conjugates and pyrrolidine alkaloids. The results suggest that bound putrescine can act as a pool for pyrrolidine alkaloid formation in systems where alkaloid biosynthesis is active. In addition, changes in arginine decarboxylase activity corresponding to increased alkaloid levels suggest a role for this enzyme in the overall biosynthesis of pyrrolidine alkaloids.

Alkaloids↗

Cytotoxicity, cell cycle kinetics and morphonuclear-induced effects of Vinca alkaloid anticancer agents.

The effects of four Vinca alkaloids (vinblastine, vincristine, vindesine and vinorelbine) on three neoplastic cell lines (the MXT mouse mammary cell line and the T24 and J82 bladder cell lines) were studied at three biological levels, i.e. cell proliferation, cell cycle kinetics and morphonuclear characteristics. These effects were studied by means of digital cell image analysis on Feulgen-stained nuclei. The aim of the present work was to characterize the effects specifically induced by Vinca alkaloids as compared with those obtained previously with other pharmacological classes of anticancer drugs. The results show that Vinca alkaloids inhibit the cell proliferation of neoplastic cell lines at a concentration of 10(-8) M except in the case of the J82 cell line, for which only a slowing down of cell proliferation was observed. Concerning the cell cycle kinetics, the results show that the Vinca alkaloids induce an accumulation of cells in the mitosis phase. This accumulation of mitotic cells was maximal after 15 h incubation in the presence of the drugs. A study of the morphonuclear-induced effects of Vinca alkaloids showed that the variance of the optical density (VOD) is strongly influenced by these Vinca alkaloids. The development of the VOD was parallel with the development of the percentage of mitosis; thus, the VOD enabled the Vinca alkaloid-induced effects to be specifically characterized from a morphonuclear point of view. On the other hand, the results show that the mean value of the variance of the optical density was very highly correlated (P < 0.001) with the efficiency of the Vinca alkaloids in terms of cytotoxicity. In clinical studies, the analysis of the development of this parameter would make it possible to assess the response to chemotherapy in the case of patients treated with Vinca alkaloids.

Adenocarcinoma↗

The effect of isoquinoline alkaloids on opiate withdrawal.

Our interest has been centered on isoquinoline alkaloids obtained from Argemone mexicana (Papaveraceae), Aristolochia constricta (Aristolochiaceae) and the opium alkaloid, papaverine. In this respect, the effect of these isoquinoline alkaloids was investigated on contractions induced by naloxone of isolated guinea pig ileum acutely exposed to morphine in vitro. The activity of these alkaloids was compared to the control compound, papaverine. Furthermore, the effect of these isoquinoline alkaloids was also determined on naloxone-precipitated withdrawal in isolated guinea pig ileum exposed to DAMGO (highly selective mu opioid receptor agonist) and U50-488H (highly selective kappa opioid receptor agonist) to test whether the possible interaction of isoquinoline alkaloids on opioid withdrawal involves mu- and/or kappa-opioid receptors. Isoquinoline alkaloids from A. mexicana (from 5 x 10(-6) to 1 x 10(-4) M), from A. constricta (1 x 10(-5) x 10(-5)-1 x 10(-4) M) as well as papaverine treatment (1 x 10(-7)-5 x 10(-6)-1 x 10(-6) M) before or after the opioid agonists were able of both preventing and reversing the naloxone-induced contraction after exposure to mu (morphine and DAMGO) or kappa (U50-488H) opiate receptor agonists in a concentration-dependent manner. Both acetylcholine response and electrical stimulation were also reduced by isoquinoline alkaloids and papaverine treatment as well as the final opiate withdrawal was still reduced. The results of the present study indicate that isoquinoline alkaloids as well as papaverine were able to produce significant influence on the opiate withdrawal in vitro and these compounds were able to exert their effects both at mu and kappa opioid agonists.

Alkaloids↗

[Study on Aconitum diterpenoid alkaloids from flowers of Aconitum kusnezoffi and its decoction by ESI-MS].

AIM: To study the change of chemical compound after decocting the flowers of Aconitum kusnzoffii Reichb (FAK) by analyzing the alkaloids in FAK and the decoction of FAK qualitatively. METHODS: The alkaloid extracts of FAK and alkaloid mixtures in the decoction of FAK were directly analyzed by electrospray ionization trap tandem mass spectrometry via by pump. RESULTS: Three novel aconitum alkaloids were found in FAK. In addition, it is found that both diester diterpenoid alkaloids and triester diterpenoid alkaloids were hydrolyzed in the decoction of FAK. The hydrolysis products of the diester-alkaloids are benzoylaconines and aconines; the hydrolysis products of the triester-alkaloids are 3-acetyl-aconines. CONCLUSION: ESI-MS" method is suitable for the analysis of Aconitum alkaloids in the decoction of FAK as well as other Aconitum plants, which offers the advantages of convenience, speed, sensitivity and specificity.

Aconitum↗

A comparison of the antimalarial activity of the cinchona alkaloids against Plasmodium falciparum in vitro.

The effects of four major cinchona alkaloids: (-) quinine, (+) quinidine, (-)cinchonidine, and (+)cinchonine against Plasmodium falciparum FCQ-27/PNG were studied. The alkaloids were tested in vitro as either single alkaloids, racemic mixtures of stereoisomers, or as an equimolar combination of all four alkaloids. Results indicate (+)quinidine to be most effective and both (+)stereoisomers were more potent than the (-)stereoisomers. Inhibitory concentrations 50% (Ki) of racemic mixtures of stereoisomers were similar to those of the (+)stereoisomers alone. The Ki of four alkaloids in equimolar combination were similar to that of the (-) cinchonidine/(+)cinchonine racemic mixture. A total alkaloidal extract of Cinchona sp. was tested and compared with the pure alkaloids. HPLC analysis indicated that (+)cinchonine, (-)cinchonidine and (-)quinine were present in a ratio of approximately 1:1:2, respectively. The total alkaloid extract, with (-)stereoisomers predominating, was less effective than the four alkaloids in combination. The nature of the interaction between stereoisomers was investigated and appears to be one of addition.

Animals↗

Application of capillary zone electrophoresis in the separation and determination of the principal gum opium alkaloids.

We describe the use of capillary zone electrophoresis (CZE) for the qualitative and quantitative determination of major alkaloids (i.e., thebaine, codeine, morphine, papavarine and narcotine) in gum opium involving the analysis of alkaloids without derivatization or purification. Three extractions with 2.5% w/v aqueous acetic acid quantitatively extracted major alkaloids. The separation was carried out by CZE using a 7:3 mixture of methanol and sodium acetate (100 mM, pH 3.1) at a potential of 15 kV, with UV detection at 224 nm. Spiking of pure reference alkaloid standards in the opium extract was used for peak identification. The influences of buffer composition, pH and voltage on the separation of alkaloids were studied. The detection limit of each alkaloid dissolved in methanol was found to be 850 ng/mL (morphine), 450 ng/mL (thebaine), 500 ng/mL (codeine), 550 ng/mL (papaverine), and 500 ng/mL (narcotine) at an injection pressure of 300 mbar (injection volume, 4 nL) with a signal-to-noise ratio of 3:1. The external standard method was used for the quantification of alkaloids. The calibration plot was based on linear regression analysis. The relative standard deviation (RSD) for peak area and migration time was in the range of 1.03-3.56% and 0.34-0.69%, respectively. Percentage compositions (g%) of opium alkaloids in five gum opium samples were found to be in the range of 14.45-15.95 (morphine), 2.0-3.45 (codeine), 1.32-2.73 (thebaine), 0.92-2.37 (papavarine), and 3.85-5.77 (narcotine). The method developed is suitable for the routine analysis of major gum opium alkaloids in samples of forensic importance.

Acetic Acid↗

Testing chemical defence based on pyrrolizidine alkaloids

Pyrrolizidine alkaloids are considered the primary defence mechanism in aposematic ithomiine butterflies and arctiid moths. Despite evidence that pyrrolizidine alkaloids are effective against some invertebrate predators, proof for a protective function of pyrrolizidine alkaloids against vertebrate predators is fragmented. The present work shows that the pyrrolizidine alkaloid monocrot-aline is unpalatable to the pileated finch, Coryphospingus pileatusand that the unpalatability is learned through association with a specific colour pattern (blue stripes). In a series of trials, using mealworms as model prey, birds rejected those to which pyrrolizidine alkaloid solution had been applied topically but accepted prey devoid of the alkaloid. Subsequent offerings of prey with pyrrolizidine alkaloid and a painted blue-striped pattern led to consistent rejections by the experimental birds. Birds were then offered blue-striped painted larvae without pyrrolizidine alkaloids ('mimics'), which were rejected at levels similar to the previous trial. The predators learned to recognize the prey as unpalatable items based on their experience in the previous encounters. These results provide evidence for the protective capacity of the pyrrolizidine alkaloid against a vertebrate predator and supports the role of these chemicals in aposematism in the Lepidoptera.Copyright 1997 The Association for the Study of Animal Behaviour1997The Association for the Study of Animal Behaviour

Journal Article↗

Distribution of norditerpene alkaloids in tall larkspur plant parts through the growing season.

Previous research showed that toxic and total alkaloid pools in tall larkspur (Delphinium barbeyi) increased during early growth, then declined precipitously during the late flower and pod stage of growth. The objective of this study was to measure the concentration and pools of toxic and total alkaloids in tall larkspur plant parts, including roots, and to evaluate the changes in these pools over the growing season as an estimate of diterpenoid alkaloid kinetics in tall larkspur. Twenty entire plants were harvested at each phenological stage: beginning of growth in the spring, early flower, early pod, late pod, and senescence. The plants were separated into their respective parts, freeze-dried, extracted, and analyzed for toxic and total alkaloid concentration, and alkaloid pools were calculated. Concentration of toxic and total alkaloids in leaves and stems declined as the plants matured, while concentration in flowers and pods increased (P < 0.004). Concentration of alkaloids in the root declined in the early growth, then increased at the end of the season (P = 0.002). Alkaloid pools in the root decreased during early growth, with a corresponding increase of pools in foliar parts. In the late flower and pod stage, alkaloid pools in the leaves and stems declined rapidly, while the pool in the crown and roots tended to increase.

Delphinium↗

ALKALOID BIOSYNTHESIS IN PLANTS: Biochemistry, Cell Biology, Molecular Regulation, and Metabolic Engineering Applications.

Recent advances in the cell, developmental, and molecular biology of alkaloid biosynthesis have heightened our appreciation for the complexity and importance of plant secondary pathways. Several biosynthetic genes involved in the formation of tropane, benzylisoquinoline, and terpenoid indole alkaloids have now been isolated. The early events of signal perception, the pathways of signal transduction, and the function of gene promoters have been studied in relation to the regulation of alkaloid metabolism. Enzymes involved in alkaloid biosynthesis are associated with diverse subcellular compartments including the cytosol, vacuole, tonoplast membrane, endoplasmic reticulum, chloroplast stroma, thylakoid membranes, and perhaps unique "biosynthetic" or transport vesicles. Localization studies have shown that sequential alkaloid biosynthetic enzymes can also occur in distinct cell types, suggesting the intercellular transport of pathway intermediates. Isolated genes have also been used to genetically alter the accumulation of specific alkaloids and other plant secondary metabolites. Metabolic modifications include increased indole alkaloid levels, altered tropane alkaloid accumulation, elevated serotonin synthesis, reduced indole glucosinolate production, redirected shikimate metabolism, and increased cell wall-bound tyramine formation. This review discusses the biochemistry, cell biology, molecular regulation, and metabolic engineering of alkaloid biosynthesis in plants.

Journal Article↗

Distribution of the fungal endophyte Neotyphodium lolii is not a major determinant of the distribution of fungal alkaloids in Lolium perenne plants.

The relationships of the distributions of the insect and mammalian mycotoxins, lolitrem B and ergovaline, and the insect-feeding deterrent, peramine, with the distribution of fungal mycelium were investigated in three genotypes of perennial ryegrass (Lolium perenne L.) infected with the endophyte Neotyphodium lolii. In planta levels and distribution of the endophyte and of the three alkaloids were assessed in parallel, and different spatial or temporal concentration gradients were observed for each. Variation in the tissue distribution of the endophyte accounted only for 20%, 6%, and 31% of the variation in ergovaline, lolitrem B, and peramine, respectively. Alkaloid-endophyte ratios, determined in individual grass tissues, showed distinct in planta distribution patterns for each alkaloid and differed in magnitude among genotypes. The ergovaline-endophyte ratio was higher in the very basal plant tissues than in the apical tissues, while the lolitrem B and peramine ratios tended to be higher in apical tissues. The lolitrem B-endophyte ratio increased with leaf age, while no consistent temporal trends were detected for the other alkaloids. The results indicate that endophyte colonisation is a minor determinant of alkaloid levels, and that accumulation of the alkaloids relative to the endophyte mycelium is affected by plant genotype and tissue in a manner specific to each alkaloid. Possible factors in the regulation of alkaloid levels in the grass plant are discussed.

Alkaloids↗

Abundant respirable ergot alkaloids from the common airborne fungus Aspergillus fumigatus.

Ergot alkaloids are mycotoxins that interact with several monoamine receptors, negatively affecting cardiovascular, nervous, reproductive, and immune systems of exposed humans and animals. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, can produce ergot alkaloids in broth culture. The objectives of this study were to determine if A. fumigatus accumulates ergot alkaloids in a respirable form in or on its conidia, to quantify ergot alkaloids associated with conidia produced on several different substrates, and to measure relevant physical properties of the conidia. We found at least four ergot alkaloids, fumigaclavine C, festuclavine, fumigaclavine A, and fumigaclavine B (in order of abundance), associated with conidia of A. fumigatus. Under environmentally relevant conditions, the total mass of ergot alkaloids often constituted >1% of the mass of the conidium. Ergot alkaloids were extracted from conidia produced on all media tested, and the greatest quantities were observed when the fungus was cultured on latex paint or cultured maize seedlings. The values for physical properties of conidia likely to affect their respirability (i.e., diameter, mass, and specific gravity) were significantly lower for A. fumigatus than for Aspergillus nidulans, Aspergillus niger, and Stachybotrys chartarum. The demonstration of relatively high concentrations of ergot alkaloids associated with conidia of A. fumigatus presents opportunities for investigations of potential contributions of the toxins to adverse health effects associated with the fungus and to aspects of the biology of the fungus that contribute to its success.

Air Microbiology↗

Effects of Uncaria tomentosa total alkaloid and its components on experimental amnesia in mice: elucidation using the passive avoidance test.

The effects of Uncaria tomentosa total alkaloid and its oxindole alkaloid components, uncarine E, uncarine C, mitraphylline, rhynchophylline and isorhynchophylline, on the impairment of retention performance caused by amnesic drugs were investigated using a step-down-type passive avoidance test in mice. In this test, the retention performance of animals treated with the amnesic and test drugs before training was assessed 24 h after training. Uncaria tomentosa total alkaloid (10-20 mg kg(-1), i.p.) and the alkaloid components (10-40 mg kg(-1), i.p.), as well as the muscarinic receptor agonist oxotremorine (0.01 mg kg(-1), i.p.), significantly attenuated the deficit in retention performance induced by the muscarinic receptor antagonist scopolamine (3 mg kg(-1), i.p.). The effective doses of uncarine C and mitraphylline were larger than those of other alkaloid components. Uncarine E (20 mg kg(-1), i.p.) also blocked the impairment of passive avoidance performance caused by the nicotinic receptor antagonist mecamylamine (15 mg kg(-1), i.p.) and the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 7.5 mg kg(-1), i.p.), but it failed to affect the deficit caused by the benzodiazepine receptor agonist diazepam (2 mg kg(-1), i.p.). Rhynchophylline significantly reduced the mecamylamine-induced deficit in passive avoidance behaviour, but it failed to attenuate the effects of CPP and diazepam. These results suggest that Uncaria tomentosa total alkaloids exert a beneficial effect on memory impairment induced by the dysfunction of cholinergic systems in the brain and that the effect of the total alkaloids is partly attributed to the oxindole alkaloids tested. Moreover, these findings raised the possibility that the glutamatergic systems are implicated in the anti-amnesic effect of uncarine E.

Alkaloids↗

Novel marine alkaloids from the tunicate Eudistoma sp. are potent regulators of cellular growth and differentiation and affect cAMP-mediated processes.

Six novel alkaloids that contain a fused tetracyclic pyrido[2,3,4-kl]acridine ring system were purified recently from the Red Sea purple tunicate Eudistoma sp. Evaluation of the effects of these alkaloids on cultured neuroblastoma and fibroblast cells revealed that they possess potent growth regulatory properties, and affect cell shape and adhesion. In mouse neuroblastoma cells, the Eudistoma alkaloids inhibited cell proliferation and induced a process of differentiation during which the cells flattened onto the surface, increased considerably in size, and extended long neurites. In hamster fibroblasts the alkaloids slowed down cell multiplication, and caused an exceptional cell flattening or elongation. In a virus-transformed derivative of the hamster fibroblasts the alkaloids restored many aspects of normal cell growth and morphology. In addition, several of the alkaloids mimicked the effects of cAMP analogs on two well-characterized cAMP-mediated processes involved in hepatic glucose metabolism--inhibition of pyruvate kinase (PK) activity and induction of mRNA for phosphoenolpyruvate carboxykinase (PEPCK). All these effects suggest that the Eudistoma alkaloids may act on the cAMP signaling system. However, a single application of these compounds was sufficient to completely block cell multiplication and to induce and sustain differentiation and "reverse transformation". Furthermore, these effects were not readily reversible following removal of the drugs. In contrast, a single application of agents that mimic or elevate cAMP induced a transient response that waned with time in culture, and the effects induced by constant elevation of cAMP reverse rapidly following drug removal. We propose that the Eudistoma alkaloids cause growth inhibition, differentiation, and reverse transformation by modifying the activity state of proteins that are involved in the regulation of cell shape and adhesion and serve as a target for the cAMP and/or other second messenger systems.

Alkaloids↗

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.

Animal Feed↗

Calystegins, a novel class of alkaloid glycosidase inhibitors.

The alkaloid extract from roots of naturally growing Convolvulus arvensis, purified by ion-exchange chromatography, showed significant inhibitory activity toward beta-glucosidase and alpha-galactosidase. The demonstrated occurrence of polyhydroxy-nortropane alkaloids, the calystegins, in C. arvensis and their structural similarity to known polyhydroxy alkaloid glycosidase inhibitors, suggested that these might be responsible for the observed activity. Pure calystegins, isolated from transformed root cultures of the related plant species Calystegia sepium, were tested for glycosidase inhibitory activity. The purity of the alkaloids was established by gas chromatography and their identity confirmed by their mass spectrometric fragmentation patterns. The trihydroxy alkaloid, calystegin A3, was a moderately good inhibitor of beta-glucosidase (Ki = 4.3 x 10(-5) M) and a weak inhibitor of alpha-galactosidase (Ki = 1.9 x 10(-4) M). An increased level of hydroxylation, as in the tetrahydroxy calystegins B, consisting of 27% calystegin B1 and 73% calystegin B2, resulted in greatly enhanced inhibitory activity. The calystegins B were potent inhibitors of beta-glucosidase (Ki = 3 x 10(-6) M) and alpha-galactosidase (Ki = 7 x 10(-6) M). These levels of activity are comparable with those of the polyhydroxy indolizidine alkaloids castanospermine and swainsonine toward alpha-glucosidase and alpha-mannosidase, respectively, and of the polyhydroxy pyrrolizidine alkaloid australine toward alpha-glucosidase. The calystegins therefore compose a new structural class of polyhydroxy alkaloids, the nortropanes, possessing potent glycosidase inhibitory properties.

Alkaloids↗

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea.

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3'-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxidoreductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway.

Alkyl and Aryl Transferases↗

Alkaloid profiles, concentration, and pools in velvet lupine (Lupinus leucophyllus) over the growing season.

Lupinus leucophyllus is one of many lupine species known to contain toxic and/or teratogenic alkaloids that can cause congenital birth defects. The concentrations of total alkaloids and the individual major alkaloids were measured in three different years from different plant parts over the phenological development of the plant. All of the alkaloids were found in the different plant tissues throughout the growing season, although their levels varied in different tissues. Concentrations of total alkaloids and the individual alkaloids varied on an annual basis and in their distribution in the different tissues. Anagyrine levels were highest in the floral tissue, lupanine and unknown F accumulated to the greatest level in the vegetative tissue, and 5,6-dehydrolupanine accumulated to the highest level in the stem. These alkaloids appear to be in a metabolically active state with the teratogenic alkaloid anagyrine accumulating to its highest level in the developing seed. The latter is, thus, the phenological stage posing the greatest danger to grazing livestock.

Alkaloids↗