Astaxanthin Extract from Haematococcus pluvialis and Its Fractions of Astaxanthin Mono- and Diesters Obtained by CCC Show Differential Antioxidant and Cytoprotective Effects on Naïve-Mouse Spleen Cells.

Carotenoids are the most abundant lipid-soluble phytochemicals and are used as dietary supplements to protect against diseases caused by oxidative stress. Astaxanthin, a xanthophyll carotenoid, is a very potent antioxidant with numerous beneficial effects on cellular functions and signaling pathways. In this study, using spleen cells from healthy Balb/c mice, we report the bio-functional effects of an astaxanthin-rich extract (EXT) prepared from the microalga Haematococcus pluvialis and its astaxanthin monoesters-rich fraction (ME) and astaxanthin diesters-rich fraction (DE) obtained by fractionation of EXT using countercurrent chromatography (CCC). After incubation under standard culture conditions (humidity, 37 °C, 5% CO2, atmospheric oxygen), the viability of untreated splenocytes, as determined by the trypan blue exclusion assay, the MTT assay, and the neutral red assay, decreases to approximately 75% after 24 h compared with naïve splenocytes. This effect correlated with the decrease in mitochondrial membrane potential and the transition of ~59% of cells to the early stage of apoptosis, as well as with the decreased ROS production, indicating that hyperoxia in cell-culture deteriorates cell functions. They are restored or stimulated by co-cultivation with EXT, ME, and DE up to 10 µg/mL in the order EXT > DE > ME, suggesting that esterification increases bioavailability to cells in vitro. ROS and H2O2 concentrations reflect mRNA transcriptional activity of Nrf2, superoxide dismutase 1 (SOD1), catalase, and glutathione peroxidase 1, as well as SOD-mediated ROS conversion, whereas they inversely correlate with iNOS-mediated NO production. The highest-tested concentration of EXT, ME, and DE (40 µg/mL) is detrimental to cells, probably because of the overwhelming scavenging activity of astaxanthin and its esters for the reactive oxygen/nitrogen species required for cellular functions and signal transduction at low physiological concentrations. In this study, we demonstrate that differential activities of ME and DE contribute to the final antioxidant and cytoprotective effects of astaxanthin extract, which is beneficial in preventing a wide range of ROS-induced adverse effects, with DE being more effective. In addition, the selection of physioxia-like conditions for pharmacological research is highlighted.

Hibberdia magna (Chrysophyceae): a promising freshwater fucoxanthin and polyunsaturated fatty acid producer.

BACKGROUND: Algae are prominent producers of carotenoids and polyunsaturated fatty acids which are greatly prized in the food and pharmaceutic industry. Fucoxanthin represents a notable high-value carotenoid produced exclusively by algae. Its benefits range far beyond just antioxidant activity and include cancer prevention, anti-diabetes, anti-obesity, and many other positive effects. Accordingly, large-scale microalgae cultivation to produce fucoxanthin and polyunsaturated fatty acids is still under intensive development in the commercial and academic sectors. Industrially exploitable strains are predominantly derived from marine species while comparable freshwater fucoxanthin producers have yet to be explored. RESULTS: In this study, we searched for freshwater fucoxanthin producers among photoautotrophic flagellates including members of the class Chrysophyceae. The initial screening turned our attention to the chrysophyte alga Hibberdia magna. We performed a comprehensive cultivation experiments using a temperature × light cross-gradient to assess the impact of these conditions on the target compounds productivity. Here we present the observations that H. magna simultaneously produces fucoxanthin (max. 1.2% dry biomass) and polyunsaturated fatty acids (max. ~ 9.9% dry biomass) and is accessible to routine cultivation in lab-scale conditions. The highest biomass yields were 3.73 g L-1 accompanied by maximal volumetric productivity of 0.54 g L-1 d-1 which are comparable values to marine microalgae fucoxanthin producers in phototrophic mode. H. magna demonstrated different optimal conditions for biomass, fucoxanthin, and fatty acid accumulation. While maximal fucoxanthin productivities were obtained in dim light and moderate temperatures (23 °C× 80 µmol m-2 s-1), the highest PUFA and overall biomass productivities were found in low temperature and high light (17-20 °C × 320-480 µmol m-2 s-1). Thus, a smart biotechnology setup should be designed to fully utilize H. magna biotechnological potential. CONCLUSIONS: Our research brings pioneer insight into the biotechnology potential of freshwater autotrophic flagellates and highlights their ability to produce high-value compounds. Freshwater fucoxanthin-producing species are of special importance as the use of sea-water-based media may increase cultivation costs and prohibits inland microalgae production.

Production of Fucoxanthin from Phaeodactylum tricornutum Using High Performance Countercurrent Chromatography Retaining Its FOXO3 Nuclear Translocation-Inducing Effect.

Phaeodactylum tricornutum is a rich source of fucoxanthin, a carotenoid with several health benefits. In the present study, high performance countercurrent chromatography (HPCCC) was used to isolate fucoxanthin from an extract of P. tricornutum. A multiple sequential injection HPCCC method was developed combining two elution modes (reverse phase and extrusion). The lower phase of a biphasic solvent system (n-heptane, ethyl acetate, ethanol and water, ratio 5/5/6/3, v/v/v/v) was used as the mobile phase, while the upper phase was the stationary phase. Ten consecutive sample injections (240 mg of extract each) were performed leading to the separation of 38 mg fucoxanthin with purity of 97% and a recovery of 98%. The process throughput was 0.189 g/h, while the efficiency per gram of fucoxanthin was 0.003 g/h. Environmental risk and general process evaluation factors were used for assessment of the developed separation method and compared with existing fucoxanthin liquid-liquid isolation methods. The isolated fucoxanthin retained its well-described ability to induce nuclear translocation of transcription factor FOXO3. Overall, the developed isolation method may represent a useful model to produce biologically active fucoxanthin from diatom biomass.

Selenium Incorporation to Amino Acids in Chlorella Cultures Grown in Phototrophic and Heterotrophic Regimes.

Microalgae accumulate bioavailable selenium-containing amino acids (Se-AAs), and these are useful as a food supplement. While this accumulation has been studied in phototrophic algal cultures, little data exists for heterotrophic cultures. We have determined the Se-AAs content, selenium/sulfur (Se/S) substitution rates, and overall Se accumulation balance in photo- and heterotrophic Chlorella cultures. Laboratory trials revealed that heterotrophic cultures tolerate Se doses ∼8-fold higher compared to phototrophic cultures, resulting in a ∼2-3-fold higher Se-AAs content. In large-scale experiments, both cultivation regimes provided comparable Se-AAs content. Outdoor phototrophic cultures accumulated up to 400 µg g-1 of total Se-AAs and exhibited a high level of Se/S substitution (5-10%) with 30-60% organic/total Se embedded in the biomass. A slightly higher content of Se-AAs and ratio of Se/S substitution was obtained for a heterotrophic culture in pilot-scale fermentors. The data presented here shows that heterotrophic Chlorella cultures provide an alternative for Se-enriched biomass production and provides information on Se-AAs content and speciation in different cultivation regimes.

Bioethanol production from microalgae polysaccharides.

The worldwide growing demand for energy permanently increases the pressure on industrial and scientific community to introduce new alternative biofuels on the global energy market. Besides the leading role of biodiesel and biogas, bioethanol receives more and more attention as first- and second-generation biofuel in the sustainable energy industry. Lately, microalgae (green algae and cyanobacteria) biomass has also remarkable potential as a feedstock for the third-generation biofuel production due to their high lipid and carbohydrate content. The third-generation bioethanol production technology can be divided into three major processing ways: (i) fermentation of pre-treated microalgae biomass, (ii) dark fermentation of reserved carbohydrates and (iii) direct "photo-fermentation" from carbon dioxide to bioethanol using light energy. All three technologies provide possible solutions, but from a practical point of view, traditional fermentation technology from microalgae biomass receives currently the most attention. This study mainly focusses on the latest advances in traditional fermentation processes including the steps of enhanced carbohydrate accumulation, biomass pre-treatment, starch and glycogen downstream processing and various fermentation approaches.

Extracellular polysaccharide produced by Chlorella vulgaris - Chemical characterization and anti-asthmatic profile.

Microalgae are the lowest plant organisms producing a wide range of metabolites that make them interesting organisms for industrial applications. Cultivation of green microalgal species Chlorella vulgaris resulted a significant production of extracellular polysaccharide (EPS). Preliminary chemico-spectroscopic studies on EPS revealed its molecular profile, a complex primary structure consisting of six monosaccharide units occurring in both furano and pyrano forms, a high sugar binding variability and the presence of partially methylated derivatives of some sugar constituents. Biological activity tests showed that EPS caused significant bronchodilatory, anti-inflammatory and antitussive effects in test animals. Chlorella EPS appears to be a promising agent for the prevention of chronic airway inflammation, which is the basic pathogenic mechanism of many respiratory diseases, including bronchial asthma.

Application of HPCCC Combined with Polymeric Resins and HPLC for the Separation of Cyclic Lipopeptides Muscotoxins A⁻C and Their Antimicrobial Activity.

Muscotoxins are cyanobacterial cyclic lipopeptides with potential applications in biomedicine and biotechnology. In this study, Desmonostoc muscorum CCALA125 strain extracts were enriched by polymeric resin treatment, and subjected to HPCCC affording three cyclic lipopeptides (1⁻3), which were further repurified by semi-preparative HPLC, affording 1, 2, and 3, with a purity of 86%, 92%, and 90%, respectively. The chemical identities of 2⁻3 were determined as muscotoxins A and B, respectively, by comparison with previously reported ESI-HRMS/MS data, whereas 1 was determined as a novel muscotoxin variant (muscotoxin C) using NMR and ESI-HRMS/MS data. Owing to the high yield (50 mg), compound 2 was broadly screened for its antimicrobial potential exhibiting a strong antifungal activity against Alternaria alternata, Monographella cucumerina, and Aspergillus fumigatus, with minimum inhibitory concentration (MIC) values of 0.58, 2.34, and 2.34 µg/mL; respectively, and weak antibacterial activity against Bacillus subtilis with a MIC value of 37.5 µg/mL. Compounds 1 and 3 were tested only against the plant pathogenic fungus Sclerotinia sclerotiorum due to their low yield, displaying a moderate antifungal activity. The developed chromatographic method proved to be an efficient tool for obtaining muscotoxins with potent antifungal properties.

The chemical profile and pharmacodynamic properties of extracellular Wollea saccata biopolymer.

Microalgae organisms are of interest for many biotechnology applications due to the production of a wide range of biologically active compounds. Incubation of Wollea saccata in a large scale afforded a mucilaginous, high molecular weight biopolymer composed of carbohydrate, protein and phenolic compounds. Sugar moiety was rich in hexoses (60%) and 6-deoxyhexoses (31%), while only 9% of pentoses was identified. Methylation analysis revealed about 40 types of methylated sugar derivatives, suggesting a very complex structure of Wollea biopolymer. Pharmacological studies revealed new pharmacodynamic properties of cyanobacteria biopolymer, i.e. antitussive and bronchodilatory. Biopolymer was able to suppress the cough reflex induced by chemical tussigen, but its effect was lower than that of codeine, the strongest antitussive agent. The bronchodilatory effect was similar or higher than the effect of salbutamol, a bronchodilatory drug used in a clinical practice. In pharmacological studies, there were no signs of toxicity or side effects in the animals following administration of Wollea biopolymer.

The cyanobacterial metabolite nocuolin a is a natural oxadiazine that triggers apoptosis in human cancer cells.

Oxadiazines are heterocyclic compounds containing N-N-O or N-N-C-O system within a six membered ring. These structures have been up to now exclusively prepared via organic synthesis. Here, we report the discovery of a natural oxadiazine nocuolin A (NoA) that has a unique structure based on 1,2,3-oxadiazine. We have identified this compound in three independent cyanobacterial strains of genera Nostoc, Nodularia, and Anabaena and recognized the putative gene clusters for NoA biosynthesis in their genomes. Its structure was characterized using a combination of NMR, HRMS and FTIR methods. The compound was first isolated as a positive hit during screening for apoptotic inducers in crude cyanobacterial extracts. We demonstrated that NoA-induced cell death has attributes of caspase-dependent apoptosis. Moreover, NoA exhibits a potent anti-proliferative activity (0.7-4.5 µM) against several human cancer lines, with p53-mutated cell lines being even more sensitive. Since cancers bearing p53 mutations are resistant to several conventional anti-cancer drugs, NoA may offer a new scaffold for the development of drugs that have the potential to target tumor cells independent of their p53 status. As no analogous type of compound was previously described in the nature, NoA establishes a novel class of bioactive secondary metabolites.

Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC.

Puwainaphycins are a recently described group of ß-amino fatty acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium (Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n-hexane-ethyl acetate-ethanol-water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min-1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate-ethanol-water (5:1:5, v/v/v) system, afforded compounds 1 and 2. The CCC separation of fraction II, with use of the n-hexane-ethyl acetate-ethanol-water (1:5:1:5, v/v/v/v) system, afforded compounds 3-7. In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min-1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1-7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC-electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1-7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1, 2, and 5) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass. Graphical Abstract Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Compounds 1 (12-hydroxy-4-methyl-Ahtea-Puw-F), 2 (11-chloro-4-methyl-Ahdoa-Puw-F), 3 (4-methyl-Ahdoa-Puw-F), 4 (4-methyl-Ahdoa-Puw-G), 5 (12-chloro-4-methyl-Ahtea-Puw-F), 6 (4-methyl-Ahtea-Puw-F) and 7 (4-methyl-Ahtea-Puw-G). Ahtea: 3-amino-2-hydroxy tetradecanoic acid. Ahdoa: 3-amino-2-hydroxy dodecanoic acid.

A liquid chromatography-mass spectrometric method for the detection of cyclic ß-amino fatty acid lipopeptides.

Bacterial lipopeptides, which contain ß-amino fatty acids, are an abundant group of bacterial secondary metabolites exhibiting antifungal and/or cytotoxic properties. Here we have developed an LC-HRMS/MS method for the selective detection of ß-amino fatty acid containing cyclic lipopeptides. The method was optimized using the lipopeptides iturin A and puwainaphycin F, which contain fatty acids of similar length but differ in the amino acid composition of the peptide cycle. Fragmentation energies of 10-55eV were used to obtain the amino acid composition of the peptide macrocycle. However, fragmentation energies of 90-130eV were used to obtain an intense fragment specific for the ß-amino fatty acid (CnH2n+2N(+)). The method allowed the number of carbons and consequently the length of the ß-amino fatty acid to be estimated. We identified 21 puwainaphycin variants differing in fatty acid chain in the crude extract of cyanobacterium Cylindrospermum alatosporum using this method. Analogously 11 iturin A variants were detected. The retention time of the lipopeptide variants showed a near perfect linear dependence (R(2)=0.9995) on the length of the fatty acid chain in linear separation gradient which simplified the detection of minor variants. We used the method to screen 240 cyanobacterial strains and identified lipopeptides from 8 strains. The HPLC-HRMS/MS method developed here provides a rapid and easy way to detecting novel variants of cyclic lipopeptides.

Cytotoxicity evaluation of large cyanobacterial strain set using selected human and murine in vitro cell models.

The production of cytotoxic molecules interfering with mammalian cells is extensively reported in cyanobacteria. These compounds may have a use in pharmacological applications; however, their potential toxicity needs to be considered. We performed cytotoxicity tests of crude cyanobacterial extracts in six cell models in order to address the frequency of cyanobacterial cytotoxicity to human cells and the level of specificity to a particular cell line. A set of more than 100 cyanobacterial crude extracts isolated from soil habitats (mainly genera Nostoc and Tolypothrix) was tested by MTT test for in vitro toxicity on the hepatic and non-hepatic human cell lines HepG2 and HeLa, and three cell systems of rodent origin: Yac-1, Sp-2 and Balb/c 3T3 fibroblasts. Furthermore, a subset of the extracts was assessed for cytotoxicity against primary cultures of human hepatocytes as a model for evaluating potential hepatotoxicity. Roughly one third of cyanobacterial extracts caused cytotoxic effects (i.e. viability<75%) on human cell lines. Despite the sensitivity differences, high correlation coefficients among the inhibition values were obtained for particular cell systems. This suggests a prevailing general cytotoxic effect of extracts and their constituents. The non-transformed immortalized fibroblasts (Balb/c 3T3) and hepatic cancer line HepG2 exhibited good correlations with primary cultures of human hepatocytes. The presence of cytotoxic fractions in strongly cytotoxic extracts was confirmed by an activity-guided HPLC fractionation, and it was demonstrated that cyanobacterial cytotoxicity is caused by a mixture of components with similar hydrophobic/hydrophilic properties. The data presented here could be used in further research into in vitro testing based on human models for the toxicological monitoring of complex cyanobacterial samples.

Separation of Aeruginosin-865 from Cultivated Soil Cyanobacterium (Nostoc sp.) by Centrifugal Partition Chromatography combined with Gel Permeation Chromatography.

Aeruginosin-865 was isolated from cultivated soil cyanobacteria using a combination of centrifugal partition chromatography (CPC) and gel permeation chromatography. The solubility of Aer-865 in different solvents was evaluated using the conductor-like screening model for real solvents (COSMO-RS). The CPC separation was performed in descending mode with a biphasic solvent system composed of water-n-BuOH-acetic acid (5:4:1, v/v/v). The upper phase was used as a stationary phase, whereas the lower phase was employed as a mobile phase at a flow rate of 10 mL/min. The revolution speed and temperature of the separation column were 1700 rpm and 25 degrees C, respectively. Preparative CPC separation followed by gel permeation chromatography was performed on 50 mg of crude extract yielding Aer-865 (3.5 mg), with a purity over 95% as determined by HPLC. The chemical identity of the isolated compound was confirmed by comparing its spectroscopic data (UV, HRESI-MS, HRESI-MS/MS) with those of an authentic standard and data available in the literature.

Cytotoxic Lipopeptide Muscotoxin A, Isolated from Soil Cyanobacterium Desmonostoc muscorum, Permeabilizes Phospholipid Membranes by Reducing Their Fluidity.

There is mounting evidence that cyanobacterial lipopeptides can kill mammalian cells, presenting a hazard to human health. Unfortunately, their mechanism of toxicity is poorly understood. We have isolated new cyclic undecalipopeptides muscotoxin A and B containing unique lipophilicresidue 3-amino-2,5-dihydroxydecanoic acid (5-OH Ahdoa). Muscotoxin B was not used for biological studies due to its poor yield. Muscotoxin A was cytotoxic to YAC-1, Sp/2, and HeLa cancer cell lines (LC(50) ranged from 9.9 to 13.2 µM after 24 h of exposure), causing membrane damage and influx of calcium ions. Subsequently, we studied this lytic mechanism using synthetic liposomes with encapsulated fluorescent probes. Muscotoxin A permeabilized liposomes composed exclusively of phospholipids, demonstrating that no proteins or carbohydrates present in biomembranes are essential for its activity. Paradoxically, the permeabilization activity of muscotoxin A was mediated by a significant reduction in membrane surface fluidity (stiffening), the opposite of that caused by synthetic detergents and cytolytic lipopeptide puwainaphycin F. At 25 °C, muscotoxin A disrupted liposomes with and without cholesterol/sphingomyelin; however, at 37 °C, it was selective against liposomes with cholesterol/sphingomyelin. It appears that both membrane fluidity and organization can affect the lytic activity of muscotoxin A. Our findings strengthen the evidence that cyanobacterial lipopeptides specifically disrupt mammalian cell membranes and bring new insights into the mechanism of this effect.

Potential and limits of Raman spectroscopy for carotenoid detection in microorganisms: implications for astrobiology.

In this paper, it is demonstrated how Raman spectroscopy can be used to detect different carotenoids as possible biomarkers in various groups of microorganisms. The question which arose from previous studies concerns the level of unambiguity of discriminating carotenoids using common Raman microspectrometers. A series of laboratory-grown microorganisms of different taxonomic affiliation was investigated, such as halophilic heterotrophic bacteria, cyanobacteria, the anoxygenic phototrophs, the non-halophilic heterotrophs as well as eukaryotes (Ochrophyta, Rhodophyta and Chlorophyta). The data presented show that Raman spectroscopy is a suitable tool to assess the presence of carotenoids of these organisms in cultures. Comparison is made with the high-performance liquid chromatography approach of analysing pigments in extracts. Direct measurements on cultures provide fast and reliable identification of the pigments. Some of the carotenoids studied are proposed as tracers for halophiles, in contrast with others which can be considered as biomarkers of other genera. The limits of application of Raman spectroscopy are discussed for a few cases where the current Raman spectroscopic approach does not allow discriminating structurally very similar carotenoids. The database reported can be used for applications in geobiology and exobiology for the detection of pigment signals in natural settings.

A hybrid non-ribosomal peptide/polyketide synthetase containing fatty-acyl ligase (FAAL) synthesizes the ß-amino fatty acid lipopeptides puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum.

A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring ß-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

Two-step separation of nostotrebin 6 from cultivated soil cyanobacterium (Nostoc sp.) by high performance countercurrent chromatography.

High performance countercurrent chromatography (HPCCC) was successfully applied for the separation of nostotrebin 6 from cultivated soil cyanobacteria in a two-step operation. A two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (4:5:4:5, v/v/v/v) was employed for the HPCCC separation. In the first-step operation, its neutral upper phase was used as stationary phase and its basic lower phase (1% NH3 in lower phase) was employed as mobile phase at a flow rate of 1 mL/min. In the second operation step, its neutral upper phase was used as stationary phase, whereas both its neutral lower phase and basic lower phase were employed as mobile phase with a linear gradient elution at a flow rate of 0.8 mL/min. The revolution speed and temperature of the separation column were 1,000 rpm and 30 °C, respectively. Using HPCCC followed by clean-up on Sephadex LH-20 gel, 4 mg of nostotrebin 6 with a purity of 99% as determined by HPLC/DAD-ESI-HRMS was obtained from 100 mg of crude extract. The chemical identity of the isolated compound was confirmed by comparing its spectroscopic data (UV, ESI-HRMS, ESI-HRMS2) with those of an authentic standard and data available in the literature.

Novel Aeruginosin-865 from Nostoc sp. as a potent anti-inflammatory agent.

Aeruginosin-865 (Aer-865), isolated from terrestrial cyanobacterium Nostoc sp. Lukesová 30/93, is the first aeruginosin-type peptide containing both a fatty acid and a carbohydrate moiety, and is the first aeruginosin to be found in the genus Nostoc. Mass spectrometry, chemical and spectroscopic analysis as well as one- and two-dimensional NMR and chiral HPLC analysis of Marfey derivatives were applied to determine the peptidic sequence: D-Hpla, D-Leu, 5-OH-Choi, Agma, with hexanoic and mannopyranosyl uronic acid moieties linked to Choi. We used an AlphaLISA assay to measure the levels of proinflammatory mediators IL-8 and ICAM-1 in hTNF-α-stimulated HLMVECs. Aer-865 showed significant reduction of both: with EC50 values of (3.5±1.5) µg mL(-1) ((4.0±1.7) µM) and (50.0±13.4) µg mL(-1) ((57.8±15.5) µM), respectively. Confocal laser scanning microscopy revealed that the anti-inflammatory effect of Aer-865 was directly associated with inhibition of NF-κB translocation to the nucleus. Moreover, Aer-865 did not show any cytotoxic effect.

A combined impedance and AlphaLISA-based approach to identify anti-inflammatory and barrier-protective compounds in human endothelium.

Chronic inflammation is at least partially mediated by the chemokine-mediated attraction and by the adhesion molecule-directed binding of leukocytes to the activated endothelium. Therefore, it is therapeutically important to identify anti-inflammatory compounds able to control the interaction between leukocytes and the endothelial compartments of the micro- and macrocirculation. When testing novel drug candidates, it is, however, of the utmost importance to detect side effects, such as potential cytotoxic and barrier-disruptive activities. Indeed, minor changes in the endothelial monolayer integrity may increase the permeability of small blood vessels and capillaries, which, in extreme cases, can lead to edema development. Here, we describe the development of a high-throughput screening (HTS) platform, based on AlphaLISA technology, able to identify anti-inflammatory nontoxic natural or synthetic compounds capable of reducing tumor necrosis factor (TNF)-induced chemokine (interleukin [IL]-8) and adhesion molecule (ICAM-1) expression in human lung microvascular endothelial cells. Quantification of cell membrane-expressed ICAM-1 and of cell culture supernatant-associated levels of IL-8 was analyzed in HTS. In parallel, we monitored monolayer integrity and endothelial cell viability using the electrical cell substrate impedance sensing method. This platform allowed us to identify natural secondary metabolites from cyanobacteria, capable of reducing ICAM-1 and IL-8 levels in TNF-activated human microvascular endothelial cells in the absence of endothelial monolayer barrier disruption.