Induction and in vitro alkaloid yield of calluses and protocorm-like bodies (PLBs) from Pinellia ternata

This study investigated the induction and in vitro alkaloid yield of calluses and protocorm-like bodies (PLBs) from Pinellia ternata (Thunb.) Berit (Araceae). We planned to use this material in future studies related to the mass production of medicinally valuable compounds and regulation of alkaloid metabolism. Different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (6-BA), kinetin (Kin), and α-naphthaleneacetic acid (NAA) were used to induce callus and PLB formation from P. ternata tuber explants. The results showed that three physiologically distinct calluses were induced by different combinations of 2,4-D, 6-BA, and Kin used in this study. The calluses differed in color, texture, differentiation status, and alkaloid content. The alkaloid content of the three calli types ranged from 0.0175% to 0.0293%. In comparison, the alkaloid content of field-grown tubers was 0.0072%. Many reports have indicated that 2,4-D suppresses the biosynthesis of secondary metabolites; however, our results show that 2,4-D promoted alkaloid production in Pinellia calluses. The combination of NAA?+?6-BA induced PLB formation. The PLB alkaloid content of 0.0321% was 1.1 to 1.8 times higher than the alkaloid content of the calluses and 4.5 times higher than the field-grown tubers. In conclusion, the induction of calluses and PLBs with alkaloid content greater than that of field-grown tubers indicates the potential use of these tissue culture materials for bioprocessing alkaloids from P. ternata and for the study of alkaloid metabolism.     

An Ergot Alkaloid Biosynthesis Gene and Clustered Hypothetical Genes from Aspergillus fumigatus

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.     

Iso-erysopinophorine,a new quaternary alkaloid from the seeds of Erythrina arborescens

The ethanolic extract of the seeds of Erythrina arborescens yielded a new quaternary alkaloid provisionally named as iso-erysopinophorine besides the other alkaloids reported previously. The new alkaloid was characterised by chemical and spectral studies.     

Na-formylechitamidine,an alkaloid from Alstonia boonei

A known alkaloid, echitamidine, and a new alkaloid, N_a-formylechitamidine, have been isolated from the stem bark of Alstonia boonei. The structure of this new alkaloid was assigned on the basis of chemical and spectroscopic data.     

Neopetrosiamine A,biologically active bis-piperidine alkaloid from the Caribbean sea sponge Neopetrosia proxima

A new tetracyclic bis-piperidine alkaloid, neopetrosiamine A (1), has been extracted from the marine sponge Neopetrosia proxima collected off the west coast of Puerto Rico. The structure of compound 1 was elucidated by analysis of spectroscopic data coupled with careful comparisons of its ¹H and ¹³C NMR data with those of a well-known 3-alkylbis-piperidine alkaloid model. The new alkaloid displayed strong in vitro cytotoxic activity against a panel of cancer cell lines as well as in vitro inhibitory activity against the pathogenic microbes Mycobacterium tuberculosis and Plasmodium falciparum.     

Production of Lysergic Acid Derivatives in Submerged Culture: IV. Inorganic Nutrition Studies with Claviceps paspali

The inorganic requirements for growth and alkaloid production by submerged cultures of a lysergic acid alkaloid-producing isolate of Claviceps paspali were determined in a defined medium. The requirement for peak growth was essentially the same as the requirement for maximal alkaloid production in the case of potassium and magnesium. With phosphorus, however, maximal growth was reached at lower levels than those required for maximal alkaloid production. Sulfur was required in smaller amounts for peak alkaloid production than for maximal growth. In the case of iron and zinc, the requirement for maximal alkaloid production was greater than for growth. Manganese and copper could be sufficiently depleted to demonstrate their essentiality for alkaloid production, but their requirements for growth could not be demonstrated.     

A new alkaloid from south african Conium species

Recent screening of South African Conium species for alkaloids as part of taxonomic studies has yielded γ-coniceine, coniine, methylconiine, conhydrine and a new alkaloid N-methylpseudoconhydrine. The relative stereochemistry of N-methylpseudoconhydrine was ascertained by ¹H NMR decoupling experiments. This latter alkaloid was found in significant amounts in the leaf and stem of some plants investigated and was the major alkaloid along with conhydrine in the leaf and stem of one group of high altitude plants. These plants also contained significant amounts of volatile oil, the major monoterpene being myrcene.     

The alkaloid content of sweet lupin seed used in feeding trials on pigs and rats

The alkaloid content of the seeds of several new cultivars of “sweet” lupins was determined. The alkaloid contents ranged from < 0.01% for L. angustifolius cv. Uniwhite to 0.09% for L. albus cv. Neuland (a). The major alkaloid component (70%) of Neuland (a) was identified as dl-lupanine. This was the sample of L. albus that markedly suppressed feed intake and growth of young pigs; alcohol extraction reduced the alkaloid content to 0.02% and the extracted lupin-seed meal supported growth and food consumption equivalent to Uniwhite.Rats given a diet containing 50% L. albus cv. Neuland (a) grew normally over 13 weeks. Rats given Neuland (a) as the sole source of protein at 10% of the diet, had a protein efficiency ratio of 0.13. Supplementation of the diet with methionine increased the p.e.r. to 2.45. The addition of increasing amounts of lupin alkaloid, up to maximum content of 0.15% of the diet, had no effect on p.e.r. The p.e.r. obtained from diets containing ethanol-extracted Neuland (a), with and without methionine, were 0.94 and 2.61 respectively, as compared to 0.36 and 2.60 for the unextracted Neuland (a) diets, suggesting that lupin alkaloids can reduce protein quality in methionine deficient diets.The conclusion is that, in marked contrast to pigs, rats are more resistant to dietary lupin alkaloids.     

Seasonal variation in the secondary chemistry of foliar and reproductive tissues of Delphinium nuttallianum

Plant secondary compounds are critical in affecting interactions between plants and their herbivores. The norditerpene alkaloids are secondary compounds in Delphinium (larkspur) species which are divided into two classes: the N-(methylsuccinimido) anthranoyllycoctonine (MSAL-type) and non MSAL-type, and are known to be toxic to herbivorous insects and livestock. Alkaloid concentrations were measured in a whole plant context in vegetative and reproductive tissues in Delphinium nuttallianum at different stages of plant maturity at two locations to explore how plant maturity affected alkaloid concentrations within a growing season. Alkaloid concentrations differed between vegetative and reproductive tissues, with vegetative tissues having significantly lower alkaloid concentrations than reproductive tissues. However, no systematic differences in alkaloid concentrations were observed at different plant maturity stages across the growing season. Based on the data we suggest that alkaloid allocation in different plant parts of D. nuttallianum is influenced by life history of the plant, consistent with plant defense theory. At one location, as pods mature the qualitative alkaloid composition changed through structural diversification of the alkaloids present. The ecological significance of this structural diversification awaits further exploration.     

Temporary immersion systems for Amaryllidaceae alkaloids biosynthesis by Pancratium maritimum L. shoot culture

The alkaloid patterns of sea daffodil (Pancratium maritimum L.) shoot culture, cultivated in a temporary immersion cultivation system were investigated. The shoots accumulated maximal amounts of biomass (0.8 g dry biomass/L and Growth Index?=?1.6) at immersion frequency with 15 min flooding and 12 h stand-by periods. At this regime P. maritimum shoots achieved the highest degree of utilization of carbon source. Twenty-two alkaloids, belonging to narciclasine, galanthamine, haemanthamine, lycorine, montanine, tazettine, homolycorine and tyramine types were identified in intracellular and extracellular alkaloid extracts. The immersion frequency affected strongly the capacity of alkaloid biosynthesis in P. maritimum shoots and at the optimum conditions of cultivation, the total intracellular alkaloid content reached up to 3,469 μg/g dry biomass. The main biosynthesized alkaloids were haemanthamine (900.1 μg/g) and lycorine (799.9 μg/g). The obtained results proved that temporary immersion technology, as a cultivation approach, and P. maritimum shoots, as a biological system, are prospective for producing wide range bioactive alkaloids.     

Combined enzymatic and chemical synthesis of N-methyllaurotetanine

The lipase of Candida cylindraceae was used to facilitate a combined enzymatic-chemical synthesis of the alkaloid, N-methyllaurotetanine. The basis for this synthesis is the regioselective enzymatic hydrolysis of the acetate ester functional group at the 2-position of diacetylboldine. Optimal esterase conditons for the yeast enzyme were established with p-nitrophenyl acetate as substrate and these were used in the hydrolysis of the alkaloid diacetate. The synthetic pathway described illustrates the value of enzymes as reagents in synthetic organic chemistry.     

Three New O-Methyltransferases Are Sufficient for All O-Methylation Reactions of Ipecac Alkaloid Biosynthesis in Root Culture of Psychotria ipecacuanha

The medicinal plant Psychotria ipecacuanha produces ipecac alkaloids, a series of monoterpenoid-isoquinoline alkaloids such as emetine and cephaeline, whose biosynthesis derives from condensation of dopamine and secologanin. Here, we identified three cDNAs, IpeOMT1–IpeOMT3, encoding ipecac alkaloid O-methyltransferases (OMTs) from P. ipecacuanha. They were coordinately transcribed with the recently identified ipecac alkaloid β-glucosidase Ipeglu1. Their amino acid sequences were closely related to each other and rather to the flavonoid OMTs than to the OMTs involved in benzylisoquinoline alkaloid biosynthesis. Characterization of the recombinant IpeOMT enzymes with integration of the enzymatic properties of the IpeGlu1 revealed that emetine biosynthesis branches off from N-deacetylisoipecoside through its 6-O-methylation by IpeOMT1, with a minor contribution by IpeOMT2, followed by deglucosylation by IpeGlu1. The 7-hydroxy group of the isoquinoline skeleton of the aglycon is methylated by IpeOMT3 prior to the formation of protoemetine that is condensed with a second dopamine molecule, followed by sequential O-methylations by IpeOMT2 and IpeOMT1 to form cephaeline and emetine, respectively. In addition to this central pathway of ipecac alkaloid biosynthesis, formation of all methyl derivatives of ipecac alkaloids in P. ipecacuanha could be explained by the enzymatic activities of IpeOMT1–IpeOMT3, indicating that they are sufficient for all O-methylation reactions of ipecac alkaloid biosynthesis.     

The alkaloid contents of sixty Nicotiana species

The alkaloid content (nicotine, nornicotine, anabasine, and anatabine) in leaves and roots of 60 Nicotiana species were analyzed by GC. All species contained alkaloids, the amounts varying with the species. There was no clearcut correlation between alkaloid amounts and subgeneric or sectional classification. The alkaloid content in the floral parts and immature and mature fruits of Nicotiana tabacum were also analyzed.     

Lupin alkaloids from sophora chrysophylla

A new lupin alkaloid, (?)-mamanine N-oxide, was isolated from Sophora chrysophylla together with 18 known alkaloids including some unusual lupin alkaloids such as kuraramine, lamprolobine, epilamprolobine, epilamprolobine N-oxide, (+)-mamanine and (?)-pohakuline. It was also shown that the alkaloid constituents of S. chrysophylla differed considerably in the leaves, stems and seeds.     

An N-formyl cyclopeptide alkaloid from the bark of zizyphus sativa

From the bark of Zizyphus sativa, previously undescribed cyclopeptide alkaloid, sativanine-F has been isolated. The structure was deduced by spectroscopic methods and chemical degradation. It is a 13-membered cyclopeptide alkaloid and provides the first example of such a naturally occurring N-formyl cyclopeptide alkaloid.     

Heterologous expression of alkaloid biosynthetic genes — a review

Tetrahydrobenzylisoquinoline alkaloids comprise a diverse class of secondary metabolites with many pharmacologically active members. The biosynthesis at the enzyme level of at least two tetrahydrobenzylisoquinoline alkaloids, the benzophenanthridine alkaloid sanguinarine in the California poppy, Eschscholtzia californica, and the bisbenzylisoquinoline alkaloid berbamunine in barberry, Berberis stolonifera, has been elucidated in detail starting from the aromatic amino acid (aa) l-tyrosine. In an initial attempt to develop alternate systems for the production of medicinally important alkaloids, one enzyme from each pathway (BBE, a covalently flavinylated enzyme of benzophenanthridine alkaloid biosynthesis and CYP80, a phenol coupling cytochrome P-450-dependent oxidase of bisbenzylisoquinoline alkaloid biosynthesis) has been purified to homogeneity, a partial aa sequence determined, and the corresponding cDNAs isolated with aid of synthetic oligos based on the aa sequences. The recombinant enzymes were actively expressed in Spodoptera frugiperda Sf9 cells using a baculovirus vector, purified and then characterized. Insect cell culture has proven to be a powerful system for the overexpression of alkaloid biosynthetic genes.     

Structure of erysophorine: a new quaternary alkaloid of Erythrina arborescens

Isolation and structure elucidation of a new quaternary indole-eryso alkaloid, named erysophorine, from seeds of Erythrina arborescens are reported. An indole-erythrina alkaloid, such as erysophorine, has not been encountered before in nature or prepared synthetically.     

(−)-3β-13α-dihydroxylupanine from cytisus scoparius

A new lupin alkaloid, (−)-3β,13α-dihydroxylupanine was isolated from Cytisus scoparius together with five known sparteine-type lupin alkaloids and tyramine. The absolute structure of the new alkaloid was confirmed by comparison of the natural product with the synthetic sample derived from (+)-13-hydroxylupanine. It was also shown that the alkaloid constituents of C. scoparius differed considerably in the aerial parts, flowers and seeds.     

Genetic variation of piperidine alkaloids in Pinus ponderosa: a common garden study

Background and Aims

Previous measurements of conifer alkaloids have revealed significant variation attributable to many sources, environmental and genetic. The present study takes a complementary and intensive, common garden approach to examine genetic variation in Pinus ponderosa var. ponderosa alkaloid production. Additionally, this study investigates the potential trade-off between seedling growth and alkaloid production, and associations between topographic/climatic variables and alkaloid production.

Methods

Piperidine alkaloids were quantified in foliage of 501 nursery seedlings grown from seed sources in west-central Washington, Oregon and California, roughly covering the western half of the native range of ponderosa pine. A nested mixed model was used to test differences among broad-scale regions and among families within regions. Alkaloid concentrations were regressed on seedling growth measurements to test metabolite allocation theory. Likewise, climate characteristics at the seed sources were also considered as explanatory variables.

Key Results

Quantitative variation from seedling to seedling was high, and regional variation exceeded variation among families. Regions along the western margin of the species range exhibited the highest alkaloid concentrations, while those further east had relatively low alkaloid levels. Qualitative variation in alkaloid profiles was low. All measures of seedling growth related negatively to alkaloid concentrations on a natural log scale; however, coefficients of determination were low. At best, annual height increment explained 19·4 % of the variation in ln(total alkaloids). Among the climate variables, temperature range showed a negative, linear association that explained 41·8 % of the variation.

Conclusions

Given the wide geographic scope of the seed sources and the uniformity of resources in the seedlings'' environment, observed differences in alkaloid concentrations are evidence for genetic regulation of alkaloid secondary metabolism in ponderosa pine. The theoretical trade-off with seedling growth appeared to be real, however slight. The climate variables provided little evidence for adaptive alkaloid variation, especially within regions.Key words: Pinus ponderosa var. ponderosa, Pinaceae, 2,6-disubstituted piperidine alkaloids, secondary products, geographic variation, progeny study, plant defense, Growth–Differentiation Balance Hypothesis, PRISM     

The ergot alkaloid gene cluster: Functional analyses and evolutionary aspects

Ergot alkaloids and their derivatives have been traditionally used as therapeutic agents in migraine, blood pressure regulation and help in childbirth and abortion. Their production in submerse culture is a long established biotechnological process. Ergot alkaloids are produced mainly by members of the genus Claviceps, with Claviceps purpurea as best investigated species concerning the biochemistry of ergot alkaloid synthesis (EAS). Genes encoding enzymes involved in EAS have been shown to be clustered; functional analyses of EAS cluster genes have allowed to assign specific functions to several gene products. Various Claviceps species differ with respect to their host specificity and their alkaloid content; comparison of the ergot alkaloid clusters in these species (and of clavine alkaloid clusters in other genera) yields interesting insights into the evolution of cluster structure. This review focuses on recently published and also yet unpublished data on the structure and evolution of the EAS gene cluster and on the function and regulation of cluster genes. These analyses have also significant biotechnological implications: the characterization of non-ribosomal peptide synthetases (NRPS) involved in the synthesis of the peptide moiety of ergopeptines opened interesting perspectives for the synthesis of ergot alkaloids; on the other hand, defined mutants could be generated producing interesting intermediates or only single peptide alkaloids (instead of the alkaloid mixtures usually produced by industrial strains).