Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.

Alkaloids from Stems of Esenbeckia leiocarpa Engl. (Rutaceae) as Potential Treatment for Alzheimer Disease

Esenbeckia leiocarpa Engl. (Rutaceae), popularly known as guaranta, goiabeira, is a native tree from Brazil. Bioactivity-guided fractionation of the ethanol stems extract afforded the isolation of six alkaloids: leiokinine A, leptomerine, kokusaginine, skimmianine, maculine and flindersiamine. All isolated compounds were tested for acetyl cholinesterase inhibition, in vitro and displayed anticholinesterasic activity. The alkaloid leptomerine showed the highest activity (IC(50) = 2.5 mu M), similar to that of the reference compound galanthamine (IC(50) = 1.7 mu M). The results showed for the first time the presence of alkaloids leptomerine and skimmianine in E. leiocarpa (Engl.) with potent anticholinesterasic activity.

Gas Chromatographic Analysis of Dimethyltryptamine and beta-Carboline Alkaloids in Ayahuasca, an Amazonian Psychoactive Plant Beverage

Introduction - Ayahuasca is obtained by infusing the pounded stems of Banisteriopsis caapi in combination with the leaves of Psychotria viridis. P. viridis is rich in the psychedelic indole N,N-dimethyltryptamine, whereas B. caapi contains substantial amounts of beta-carboline alkaloids, mainly harmine, harmaline and tetrahydroharmine, which are monoamine-oxidase inhibitors. Because of differences in composition in ayahuasca preparations, a method to measure their main active constituents is needed. Objective - To develop a gas chromatographic method for the simultaneous determination of dimethyltryptamine and the main beta-carbolines found in ayahuasca preparations. Methodology - The alkaloids were extracted by means of solid phase extraction (C(18)) and detected by gas chromatography with nitrogen/phosphorous detector. Results - The lower limit of quantification (LLOQ) was 0.02 mg/mL for all analytes. The calibration curves were linear over a concentration range of 0.02-4.0 mg/mL (r(2) > 0.99). The method was also precise (RSD < 10%). Conclusion - A simple gas chromatographic method to determine the main alkaloids found in ayahuasca was developed and validated. The method can be useful to estimate administered doses in animals and humans for further pharmacological and toxicological investigations of ayahuasca. Copyright (C) 2009 John Wiley & Sons, Ltd.

The antitumoral, trypanocidal and antileishmanial activities of extract and alkaloids isolated from Duguetia furfuracea

The alkaloid extract and five alkaloids isolated from subterranean stem bark of Duguetia furfuracea (Annonaceae) were investigated for the following activities: antitumoral, trypanocidal and leishmanicidal. Dicentrinone showed weak cytotoxicity, but it had the strongest leishmanicidal activity IC(50) 0.01 mu M). Duguetine and duguetine beta-N-oxide caused considerable antitumoral activity in every cell lines evaluated, although duguetine was more active against trypomastigote forms (IC(50) 9.32 mu M) than other alkaloids tested. (C) 2009 Elsevier GmbH. All rights reserved.

The haliclonacyclamines, cytotoxic tertiary alkaloids from the tropical marine sponge Haliclona sp

2D-NMR spectroscopic data is reported for the haliclonacyclamines A - D (1)-(4) and for two bismethiodide adducts (5) and (6). The structures of two new alkaloids, haliclonacyclamines C (3) and D (4), which are the 15,16-dihydro analogues of the haliclonacyclamines A (1) and B (2) are described. Revised assignments deduced by 2D-INADEQUATE spectroscopy are presented for (1) and (2). The alkene substituent in the C,, spacer group of (2) and (4) is positioned between C27-C28 by NMR, and confirmed by x-ray structural analysis for (2). Metabolite (3) has a C25-C26 double bond. (C) 1998 Elsevier Science Ltd. All rights reserved.

A sponge/dinoflagellate association in the haplosclerid sponge Haliclona sp.: cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation

Light-microscopic and electron-microscopic studies of the tropical marine sponge Haliclona sp. (Or der: Haplosclerida Family: Haliclonidae) from Heron Island, Great Barrier Reef, have revealed that this sponge is characterized by the presence of dinoflagellates and by nematocysts. The dinoflagellates are 7-10 mu m in size, intracellular, and contain a pyrenoid with a single stalk, whereas the single chloroplast is branched, curved, and lacks grana. Mitochondria are present, and the nucleus is oval and has distinct chromosomal structure. The dinoflagellates are morphologically similar to Symbiodinium microadriaticum, the common intracellular symbiont of corals, although more detailed biochemical and molecular studies are required to provide a precise taxonomic assignment. The major sponge cell types found in Haliclona sp, are spongocytes, choanocytes, and archaeocytes; groups of dinoflagellates are enclosed within large vacuoles in the archaeocytes. The occurrence of dinoflagellates in marine sponges has previously been thought to be restricted to a small group of sponges including the excavating hadromerid sponges; the dinoflagellates in these sponges are usually referred to as symbionts. The role of the dinoflagellates present in Haliclona sp. as a genuine symbiotic partner requires experimental investigation. The sponge grows on coral substrates, from which it may acquire the nematocysts, and shows features, such as mucus production, which are typical of some excavating sponges. The cytotoxic alkaloids, haliclonacyclamines A and B, associated with Haliclona sp. are shown by Percoll density gradient fractionation to be localized within the sponge cells rather than the dinoflagellates. The ability to synthesize bioactive compounds such as the haliclonacyclamines may help Haliclona sp. to preserve its remarkable ecological niche.

Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11-alpha-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae)

Neural mechanisms underlying the onset and maintenance of epileptic seizures involve alterations in inhibitory and/or excitatory neurotransmitter pathways. Thus, the prospecting of novel molecules from natural products that target both inhibition and excitation systems has deserved interest in the rational design of new anticonvulsants. We isolated the alkaloids (+)-erythravine and ( +)-11-alpha-hydroxyerythravine from the flowers of Erythrina mulungu and evaluated the action of these compounds against chemically induced seizures in rats. Our results showed that the administration of different doses of (+)-erythravine inhibited seizures evoked by bicuculline, pentylenetetrazole, and kainic acid at maximum of 80, 100, and 100%, respectively, whereas different doses of (+)-11-alpha-hydroxy-erythravine inhibited seizures at a maximum of 100% when induced by bicuculline, NMDA, and kainic acid, and, to a lesser extent, PTZ (60%). The analysis of mean latency to seizure onset of nonprotected animals, for specific doses of alkaloids, showed that (+)-erythravine increased latencies to seizures induced by bicuculline. Although (+)-erythravine exhibited very weak anticonvulsant action against seizures induced by NMDA, this alkaloid increased the latency in this assay. The increase in latency to onset of seizures promoted by (+)-11-alpha-hydroxy-erythravine reached a maximum of threefold in the bicuculline test. All animals were protected against death when treated with different doses of (+)-11-alpha-hydroxy-erythravine in the tests using the four chemical convulsants. Identical results were obtained when using (+)-erythravine in the tests of bicuculline, NMDA, and VIZ, and, to a lesser extent, kainic acid. Therefore, these data validate the anticonvulsant properties of the tested alkaloids, which is of relevance in consideration of the ethnopharmacological/biotechnological potential of E. mulungu. (C) 2010 Elsevier Inc. All rights reserved.

Antimicrobial Activities of Indole Alkaloids from Tabernaemontana catharinensis

Tabernaemontana catharinensis root bark ethanol extract, EB2 fraction and the MMV alkaloid (12-methoxy-4-methylvoachalotine) were evaluated for their antimicrobial activities. T. catharinensis ethanol extract was effective against both strains of the dermatophyte Trichophyton rubrum at concentrations of 2.5 mg/mL (wild strain) and 1.25 mg/mL (mutant strain), while the EB2 fraction and MMV alkaloid showed a strong antifungal activity against wild and mutant strains with MIC values of <0.02 and 0.16 mg/mL, respectively. The EB2 fraction showed a strong antibacterial activity against ATCC strains of S. aureus, S. epidermidis, E. coli and P. aeruginosa with MICs from <0.02 to 0.04 mg/mL, as well as against resistant clinical isolates species of Enterococcus sp, Klebsiella oxytoca, Citrobacter, K. pneumoniae, P. mirabilis, S. aureus, S. epidermidis, E. coli and P. aeruginosa with MIC values ranging from 0.04 to 0.08 mg/mL. The MMV alkaloid presented a MIC of 0.16 mg/mL against the strains of S. aureus and E. coli ATCC. For the resistant clinical isolates Enterococcus sp, Citrobacter, S. aureus, S. epidermidis, E. coil and P. aeruginosa the MIC of MMV ranged from 0.08 to 0.31 mg/mL. The chromatography analysis of the EB2 fraction revealed the presence of indole alkaloids, including MMV, possibly responsible for the observed antimicrobial activity.

New chlorinated peptides from the tropical marine sponge Dysidea sp.

Five new chlorinated peptides (5)-(9) have been isolated from a Dysidea sp. and identified by two-dimensional NMR spectroscopy. The absolute stereochemistry of the metabolites was deduced by chemical correlation with S-(-)-4,4,4-trichloro-3-methylbutanoic acid (10) and with an alcohol (11). (C) 2001 Elsevier Science Ltd. All rights reserved.

Characterisation of alkaloids from some Australian Stephania (Menispermaceae) species

Chemical investigations of some Stephania species native to Australia and reportedly employed by Aboriginal people as therapeutic agents. are described. The alkaloids from the forest vines Stephania bancroftii F.M. Bailey and S. aculeata F.M. Bailey (Menispermaceae) have been isolated and characterised. The major alkaloids in the tuber of the former species are (-)-tetra-hydropalmatine and (-)-stephanine, whereas these are minor components in the leaves, from which a C-7 hydroxylated aporphine has been identified. The major tuber alkaloids in S. aculcata are (+)-laudanidine, and the morphinoid, (-)-amurine, whose absolute stereochemistry has been established by X-ray structural analysis of the methiodide derivative. No significant levels of alkaloids were detected in S. japonica. Complete and unambiguous H-1 and C-13 NMR data are presented for these alkaloids. (C) 2003 Elsevier Ltd. All rights reserved.

Bridgehead arylation: A direct route to advanced intermediates for the synthesis of C-20 diterpene alkaloids

[GRAPHICS] Rapid access to the ABCE ring system of the C-20 diterpene alkaloids was achieved by silver(I)-promoted intramolecular Friedel-Crafts arylation of a functional group-specific 5-bromo-3-azabicyclo[3.3.1]nonane derivative.

Development and validation of a novel method for the analysis of chlorinated pesticides in soils using microwave-assisted extraction–headspace solid phase microextraction and gas chromatography–tandem mass spectrometry

A new procedure for determining eleven organochlorine pesticides in soils using microwave-assisted extraction (MAE) and headspace solid phase microextraction (HS-SPME) is described. The studied pesticides consisted of mirex, α- and γ-chlordane, p,p’-DDT, heptachlor, heptachlor epoxide isomer A, γ-hexachlorocyclohexane, dieldrin, endrin, aldrine and hexachlorobenzene. The HS-SPME was optimized for the most important parameters such as extraction time, sample volume and temperature. The present analytical procedure requires a reduced volume of organic solvents and avoids the need for extract clean-up steps. For optimized conditions the limits of detection for the method ranged from 0.02 to 3.6 ng/g, intermediate precision ranged from 14 to 36% (as CV%), and the recovery from 8 up to 51%. The proposed methodology can be used in the rapid screening of soil for the presence of the selected pesticides, and was applied to landfill soil samples.

Biosynthesis-Assisted Structural Elucidation of the Bartolosides, Chlorinated Aromatic Glycolipids from Cyanobacteria

The isolation of the bartolosides, unprecedented cyanobacterial glycolipids featuring aliphatic chains with chlorine substituents and C-glycosyl moieties, is reported. Their chlorinated dialkylresorcinol (DAR) core presented a major structural-elucidation challenge. To overcome this, we discovered the bartoloside (brt) biosynthetic gene cluster and linked it to the natural products through in vitro characterization of the DAR-forming ketosynthase and aromatase. Bioinformatic analysis also revealed a novel potential halogenase. Knowledge of the bartoloside biosynthesis constrained the DAR core structure by defining key pathway intermediates, ultimately allowing us to determine the full structures of the bartolosides. This work illustrates the power of genomics to enable the use of biosynthetic information for structure elucidation.

Synthesis and antibiotic activity of some β-Carboline alkaloids

Nine β-carboline alkaloids were synthetized and screened for antibiotic activity. Six of the compounds testes showed inhibitory activity against one or more of the microorganisms assayed.

Chlorite Dismutase from perchlorate reducing bacteria. A potential biocatalyst to eliminate chlorinated species from polluted water resources and industrial wastes

Magnetospirillum (M.) sp. strain Lusitani, a perchlorate reducing bacteria (PRB), was previously isolated from a wastewater treatment plant and phylogenetic analysis was performed to classify the isolate. The DNA sequence of the genes responsible for perchlorate reduction and chlorite dismutation was determined and a model was designed based on the physiological roles of the proteins involved in the pcr-cld regulon. Chlorite dismutase (Cld) was purified from Magnetospirillum sp. strain Lusitani cells grown in anaerobiosis in the presence of perchlorate. The protein was purified up to electrophoretic grade using HPLC techniques as a 140 kDa homopentamer comprising five ~28 kDa monomers. Steady-state kinetic studies showed that the enzyme follows a Michaelis-Menten model with optimal pH and temperature of 6.0 and 5°C, respectively. The average values for the kinetic constants KM and Vmax were respectively 0.56 mM and 10.2 U, which correspond to a specific activity of 35470 U/mg and a turnover number of 16552 s-1. Cld from M. sp. strain Lusitani is inhibited by the product chloride, but not by dioxygen. Inhibition constants KiC= 460 mM and KiU= 480 mM indicated that sodium chloride is a weak mixed inhibitor of Cld, with a slightly stronger competitive character. The X-ray crystallography structure of M. sp. strain Lusitani Cld was solved at 3.0 Å resolution. In agreement with cofactor content biochemical analysis, the X-ray data showed that each Cld monomer harbors one heme b coordinated by a histidine residue (His188), hydrogen-bonded to a conserved glutamic acid residue (Glu238). The conserved neighboring arginine residue (Arg201) important for substrate positioning, was found in two different conformations in different monomers depending on the presence of the exogenous ligand thiocyanate. UV-Visible and CW-EPR spectroscopies were used to study the effect of redox agents, pH and exogenous ligands on the heme environment.