Antiviral Activity and Underlying Action Mechanism of Euglena Extract against Influenza Virus.

It is difficult to match annual vaccines against the exact influenza strain that is spreading in any given flu season. Owing to the emergence of drug-resistant viral strains, new approaches for treating influenza are needed. Euglena gracilis (hereinafter Euglena), microalga, used as functional foods and supplements, have been shown to alleviate symptoms of influenza virus infection in mice. However, the mechanism underlying the inhibitory action of microalgae against the influenza virus is unknown. Here, we aimed to study the antiviral activity of Euglena extract against the influenza virus and the underlying action mechanism using Madin-Darby canine kidney (MDCK) cells. Euglena extract strongly inhibited infection by all influenza virus strains examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. A time-of-addition assay revealed that Euglena extract did not affect the cycle of virus replication, and cell pretreatment or prolonged treatment of infected cells reduced the virus titer. Thus, Euglena extract may activate the host cell defense mechanisms, rather than directly acting on the influenza virus. Moreover, various minerals, mainly zinc, in Euglena extract were found to be involved in the antiviral activity of the extract. In conclusion, Euglena extract could be a potent agent for preventing and treating influenza.

Euglena Species: Bioactive Compounds and their Varied Applications.

Euglena, a microalga, has gained a great attention as it contains several bioactive compounds including food supplements, drugs and biofuels. The genus Euglena includes >300 species of unicellular, fresh water flagellates. The objective of this review article concerns the presentation of updated information on pharmacological and therapeutic properties and industrial implications of molecules isolated from Euglena species. A bibliographic search of scientific literature published till March, 2020 was made from scientific databases using different search engines. Euglena produces several antioxidant molecules, such as ß-carotene, L-ascorbic acid, polymers of unsaturated fatty acids and phytotoxins useful in manufacturing many pharmaceutical, cosmetics, and nutraceutical compounds. It is a rich source of antimicrobial, anticancer, immunomodulatory compounds. Though, several studies have indicated its therapeutic applications, extensive research is needed to explore its efficacy against many pathophysiological conditions including toxicity assessment of compound(s). Nevertheless, the biotechnological influence on industrial production of Euglena has been less exploited.

Exploring the Microalga Euglena cantabrica by Pressurized Liquid Extraction to Obtain Bioactive Compounds.

In the present study, the chemical composition of the microalga Euglena cantabrica was investigated. The extraction of bioactive compounds was done using pressurized liquid extraction (PLE) at different temperatures (40-180 °C) and using green solvents (ethanol-water mixtures). A statistical design of experiments was used to optimize the maximum antioxidant capacity of the extracts by response surface methodology. The antioxidant capacity was determined through the inhibition of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, while the chemical analyses of the extracts were carried out using different chromatographic techniques. Chlorophylls and carotenoids were analyzed by high-performance liquid chromatography coupled to a diode array detector and mass spectrometry (HPLC-DAD-MS/MS) and carbohydrates by gas chromatography with flame ionization detection (GC-FID) and high-pressure size-exclusion chromatography coupled to an evaporative light-scattering detector (HPSEC-ELSD). The results showed different possibilities for the extraction conditions, depending on the desired bioactivity or chemical composition. Briefly, (i) mixtures of ethanol-water containing around 40% ethanol at 180 °C gave the best antioxidant capacity, (ii) mixtures containing around 50% ethanol at 110 °C gave the best yield of ß-glucan paramylon, and (iii) the use of pure ethanol at a low temperature (40 °C) is the best choice for the recovery of carotenoids such as diatoxanthin. Summing up, E. cantabrica seems to be a good candidate to be used in biorefinery to obtain different bioactive compounds.

Dietary supplement product composed of natural ingredients as a suspected cause of erythema multiforme: A case report and identification for the confident false positivity of lymphocyte transformation test.

Growing and sustainable consumption of health-care products raises a controversial issue underlying the reliability of an in vitro diagnostic approach for adverse skin reaction. This report aimed to: (i) discuss the causative nature of a commercial dietary supplement composed of natural ingredients, particularly an Euglena-containing product, suspicious for erythema multiforme in our exemplified case; and (ii) to address the assay suitability of the lymphocyte transformation test (LTT) for identifying allergic reaction to any ingredient(s) of the product. A Japanese elderly man developed erythema multiforme after intake of a commercially available natural dietary product, whose LTT was positive. His clinical course and positive LTT suggested a provisional diagnosis of natural dietary product-induced eruption. We conducted an inquiry survey for the standard LTT with any commercial products containing Euglena in three major Japanese laboratory services and identified 22 subjects, almost all of whom (21/22, 95.6%) showed a positive LTT for any Euglena-containing products as a suspected causative. Seven normal healthy volunteers who had no intake history of Euglena-containing products showed an equivalent LTT positivity rate with the same product taken by our case; culprit components of the product included Euglena, Angelica keiskei, Barley grass and Chlorella. A cell-free culture system and enzyme-linked immunoassay suggest that the high LTT positivity relies on the non-specific lymphoproliferative activity, and not contamination of uncharacterized microorganisms and endotoxins. Because of the constitutive false positivity of LTT, this assay is unreliable for in vitro supportive diagnosis of adverse skin events caused by dietary products containing particular natural ingredients, as well as herbal materials.

Algae through the looking glass.

Microalgae are one of the most suitable subjects for testing the potentiality of light microscopy and image analysis, because of the size of single cells, their endogenous chromaticity, and their metabolic and physiological characteristics. Microscope observations and image analysis can use microalgal cells from lab cultures or collected from water bodies as model to investigate metabolic processes, behavior/reaction of cells under chemical or photic stimuli, and dynamics of population in the natural environment in response to changing conditions. In this paper we will describe the original microscope we set up together with the image processing techniques we improved to deal with these topics. Our system detects and recognizes in-focus cells, extracts their features, measures cell concentration in multi-algal samples, reconstructs swimming cell tracks, monitors metabolic processes, and measure absorption and fluorescent spectra of subcellular compartments. It can be used as digital microscopy station for algal cell biology and behavioral studies, and field analysis applications.

Synthesis of nanofiber-formable carboxymethylated Euglena-derived ß-1,3-glucan.

Carboxymethylation of paramylon, a storage polysaccharide of Euglena, was explored using homogeneous and heterogeneous reaction systems. Homogeneous reactions yielded carboxymethyl paramylons with a low degree of substitution of carboxymethyl group (DScm) of ∼0.13. Heterogeneous reactions of paramylon with chloroacetic acid in a mixture of 2-propanol and a NaOH aqueous solution yielded carboxymethyl paramylons with a high DScm of up to ∼0.80. An essential step for successful carboxymethylation using the heterogeneous reaction systems devised in this study was the pretreatment of a paramylon particle to break its inherent highly crystalline state into an amorphous one. A high degree of substitution provided carboxymethyl paramylons with a nanofiber-forming ability in the aqueous phase while a low degree provided them with a fiber-forming ability in both the aqueous and solid phases.

Molecular characterization of phytoplankton dissolved organic matter (DOM) and sulfur components using high resolution Orbitrap mass spectrometry.

Orbitrap high resolution mass spectrometry (HRMS) with electrospray ionization in both positive and negative polarity was conducted on Suwannee River fulvic acid (SRFA), Pony Lake fulvic acid (PLFA) standards, and dissolved organic matter (DOM) released by freshwater phytoplankton (Scenedesmus obliquus, Euglena mutabilis, and Euglena gracilis). Three-dimensional van Krevelen diagrams expressing various oxygenation states of sulfur molecules and abundance plots of sulfur-containing species were constructed. Orbitrap HRMS analysis of SRFA found a high density of peaks in the lignin region (77 %) and low density of protein material (6.53 %), whereas for PLFA, 25 % of the total peaks were lignin related compared to 56 % of peaks in protein regions, comparable with other HRMS studies. Phytoplankton-derived DOM of S. obliquus, E. mutabilis, and E. gracilis was dominated by protein molecules at respective percentages of 36, 46, and 49 %, and is consistent with previous experiments examining phytoplankton-derived DOM composition. The normalized percentage of SO-containing compounds was determined among the three phytoplankton to be 56 % for Scenedesmus, 54 % for E. mutabilis, and 47 % for E. gracilis, suggesting variation between sulfur content in phytoplankton-derived DOM and differences in metal binding capacities. These results suggest the level of resolution by Orbitrap mass spectrometry is sufficient for preliminary characterization of phytoplankton DOM at an affordable cost relative to other HRMS techniques.

Separation and Identification of Odd Chain Triacylglycerols of the Protozoan Khawkinea quartana and the Mold Mortierella alpina Using LC-MS.

Liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-MS/APCI) with reversed- and chiral phases was used for separation of triacylglycerols (TAG) from protozoan and mold. This study describes the separation and identification of odd numbered chains of regioisomers and enantiomers of triacylglycerols from different natural sources, i.e., the protozoan Khawkinea quartana and the mold Mortierella alpina. Using the above-mentioned separation methods and the synthesis of appropriate standards of TAG, we identified regioisomers and enantiomers of both even and odd numbered TAG. The biosynthesis of odd numbered TAG was found to be strictly stereospecific and to depend on the production microorganism, one enantiomer predominating in the protozoan and the other in the mold. It was proved that even numbered TAG are synthesized in a higher optical purity, which can be explained by a higher affinity of acyltransferases to the respective substrate, i.e., to even chain PUFA.

Phytochemical profile of a microalgae Euglena tuba and its hepatoprotective effect against iron-induced liver damage in Swiss albino mice.

AIMS: This study was aimed to evaluate different phytochemical constituents and the ameliorating effect of 70% methanol extract of Euglena tuba (ETME) on iron overload-induced liver injury, along with its in vitro iron-chelating and DNA protection effects. METHODS AND RESULTS: Phytochemicals of ETME were identified by GC-MS analysis. Iron chelation and protection of Fenton reaction-induced DNA damage was conducted in vitro. Post oral administration of ETME to iron-overloaded mice, the levels of serum parameters, antioxidant enzymes, liver iron, lipid peroxidation, protein carbonyl and hydroxyproline contents were measured. ETME showed inhibition of lipid peroxidation, protein oxidation and liver fibrosis. The serum markers and liver iron were lessened, whereas enhanced levels of liver antioxidant enzymes were detected in ETME-treated group. Furthermore, the histopathological observations also substantiated the protective effects of the extract. CONCLUSIONS: Several bioactive compounds identified by GC-MS may be the basis of hepatoprotective as well as antioxidant and iron-chelating effect of ETME. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently available iron-chelating agents show several side effects and limitations which may be overcome by ETME, which suggest its benefit against pathology of iron overload-linked diseases. Hence, ETME can be used as a promising hepatoprotective agent.

Assessment of the phytochemical constituents and antioxidant activity of a bloom forming microalgae Euglena tuba.

BACKGROUND: Unstable generation of free radicals in the body are responsible for many degenerative diseases. A bloom forming algae Euglena tuba growing abundantly in the aquatic habitats of Cachar district in the state of Assam in North-East India was analysed for its phytochemical contents, antioxidant activity as well as free radical scavenging potentials. RESULTS: Based on the ability of the extract in ABTS•+ radical cation inhibition and Fe3+ reducing power, the obtained results revealed the prominent antioxidant activity of the algae, with high correlation coefficient of its TEAC values to the respective phenolic and flavonoid contents. The extract had shown its scavenging activity for different free radicals and 41.89 ± 0.41 µg/ml, 5.83 ± 0.07 µg/ml, 278.46 ± 15.02 µg/ml and 223.25 ± 4.19 µg/ml were determined as the IC50 values for hydroxyl, superoxide, nitric oxide and hypochlorous acid respectively, which are lower than that of the corresponding reference standards. The phytochemical analysis also revealed that the phenolics, flavonoids, alkaloids, tannins and carbohydrates are present in adequate amount in the extract which was confirmed by HPLC analysis. CONCLUSIONS: The results showed that 70% methanol extract of the algae possesses excellent antioxidant and free radical scavenging properties.

Assessment of the phytochemical constituents and antioxidant activity of a bloom forming microalgae Euglena tuba

BACKGROUND: Unstable generation of free radicals in the body are responsible for many degenerative diseases. A bloom forming algae Euglena tuba growing abundantly in the aquatic habitats of Cachar district in the state of Assam in North-East India was analysed for its phytochemical contents, antioxidant activity as well as free radical scavenging potentials. RESULTS: Based on the ability of the extract in ABTS•+ radical cation inhibition and Fe3+ reducing power, the obtained results revealed the prominent antioxidant activity of the algae, with high correlation coefficient of its TEAC values to the respective phenolic and flavonoid contents. The extract had shown its scavenging activity for different free radicals and 41.89 ± 0.41 µg/ml, 5.83 ± 0.07 µg/ml, 278.46 ± 15.02 µg/ml and 223.25 ± 4.19 µg/ml were determined as the IC50 values for hydroxyl, superoxide, nitric oxide and hypochlorous acid respectively, which are lower than that of the corresponding reference standards. The phytochemical analysis also revealed that the phenolics, flavonoids, alkaloids, tannins and carbohydrates are present in adequate amount in the extract which was confirmed by HPLC analysis. CONCLUSIONS: The results showed that 70% methanol extract of the algae possesses excellent antioxidant and free radical scavenging properties.

Quantitative mass spectrometric analysis and post-extraction stability assessment of the euglenoid toxin euglenophycin.

Euglenophycin is a recently discovered toxin produced by at least one species of euglenoid algae. The toxin has been responsible for several fish mortality events. To facilitate the identification and monitoring of euglenophycin in freshwater ponds, we have developed a specific mass spectrometric method for the identification and quantitation of euglenophycin. The post-extraction stability of the toxin was assessed under various conditions. Euglenophycin was most stable at room temperature. At 8 °C there was a small, but statistically significant, loss in toxin after one day. These methods and knowledge of the toxin's stability will facilitate identification of the toxin as a causative agent in fish kills and determination of the toxin's distribution in the organs of exposed fish.

Preparation of carboxylic acid-bearing polysaccharide nanofiber made from euglenoid ß-1,3-glucans.

This paper introduces a new strategy for creating surface modified polysaccharide nanofibers. To demonstrate proof of principle, the synthesis, structure, and self-assembly behavior of a carboxylic acid-bearing polysaccharide made from paramylon (ß-1,3-glucan) and succinic anhydride were investigated. Examination by a combination of NMR, FT-IR, and SEC-MALLS confirmed that successful preparation of the desired succinylated paramylon without significant depolymerization. NMR, SEC-MALLS, visible absorption and CD spectroscopic analyses indicated that the paramylon derivative forms the triplex structure in solutions. SEM observation revealed that succinylated paramylon forms a nanofiber that has carboxylic acid on the surface.

Surface properties and intracellular speciation revealed an original adaptive mechanism to arsenic in the acid mine drainage bio-indicator Euglena mutabilis.

Euglena mutabilis is a protist ubiquitously found in extreme environments such as acid mine drainages which are often rich in arsenic. The response of E. mutabilis to this metalloid was compared to that of Euglena gracilis, a protist not found in such environments. Membrane fatty acid composition, cell surface properties, arsenic accumulation kinetics, and intracellular arsenic speciation were determined. The results revealed a modification in fatty acid composition leading to an increased membrane fluidity in both Euglena species under sublethal arsenic concentrations exposure. This increased membrane fluidity correlated to an induced gliding motility observed in E. mutabilis in the presence of this metalloid but did not affect the flagellar dependent motility of E. gracilis. Moreover, when compared to E. gracilis, E. mutabilis showed highly hydrophobic cell surface properties and a higher tolerance to water-soluble arsenical compounds but not to hydrophobic ones. Finally, E. mutabilis showed a lower accumulation of total arsenic in the intracellular compartment and an absence of arsenic methylated species in contrast to E. gracilis. Taken together, our results revealed the existence of a specific arsenical response of E. mutabilis that may play a role in its hypertolerance to this toxic metalloid.

Identification of euglenophycin--a toxin found in certain euglenoids.

Currently cyanoprokaryotic algae, diatoms, haptophytes, dinoflagellates, euglenoids, and rhaphidophytes are known to produce algal toxins. A previous study by the authors reported euglenoid algae producing toxin(s) in aquaculture ponds, with confirmation based on positive fish bioassays following exposure to the isolated clonal algal cultures. Toxicity was observed in euglenoid culture isolates obtained from the pond as well as a clonal, culture collection taxon. Here we provide conclusive evidence for euglenoid toxin production, including HPLC/MS, MS/MS, and NMR analyses of a clonal (non-axenic) isolate of Euglena sanguinea grown in batch culture. Following wet chemical serial fractionation, toxic activity was identified in both the methanol and hexane extracts. These extracts were then purified using HPLC. Bioassay-guided HPLC fractionation of these two extracts demonstrated that a single class of toxic compounds, identical in mass and similar in molecular structure, was produced by this organism. The toxic compounds exhibited a maximal UV absorbance at 238nm and gave diagnostic mass peaks at 306 (MH(+)) and 288 (MH(+)-H(2)O). Unambiguous molecular structural determination was carried out by high field NMR analysis operating in 1- and 2-dimensions. Though a predominant isomer represented the bulk of the toxin, several stereo- and structural isomers were evidenced by NMR, and HPLC/MS. This compound is an alkaloid similar in structure to fire ant venom. The compound exhibits ichthyotoxic, herbicidal and anticancer activity at low ppm to ppb dosages.

A new method for the identification and the structural characterisation of carotenoid esters in freshwater microorganisms by liquid chromatography/electrospray ionisation tandem mass spectrometry.

Liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) has been employed to identify carotenoid esters present in raw organic extracts of pigmented freshwater microalgae and to gain structural information on these compounds. In particular, acyl carotenoid derivatives of Haematococcus pluvialis and Euglena sanguinea have been characterised by tandem mass spectrometry (MS/MS) in a quadrupole ion trap. ESI-MS/MS allows recognition of the presence of carotenoid esters in complicated mixtures without any initial chromatographic work-up and without the need to use UV-Vis photo-diode array (PDA) detectors. Product ion scans of the [M + Na]+ ion lead to known neutral losses of the C7H8 and C8H10 residues from the conjugated polyene moiety of the carotenoid unit, that permit the unambiguous identification of the carotenoid itself. These structurally relevant ions are not observed in positive or negative ion APCI (atmospheric pressure chemical ionisation) mass spectra. Moreover, the several product ions observed in positive and/or negative ion ESI-MS/MS not only are a diagnostic signature of the main structural features of the acyl chains such as length, position and unsaturation, but also display the nominal mass of the parent xanthophyll. Our methodology has been validated (i) by using esters of astaxanthin obtained from off-line purification of the H. pluvialis extracts and structurally elucidated through proton nuclear magnetic resonance (1H-NMR) spectroscopy and (ii) by product analysis of esters by alkaline hydrolysis. The characterisation of the unknown carotenoid esters of E. sanguinea is a demonstration of the capabilities of this methodology.

Functional transplant of photoactivated adenylyl cyclase (PAC) into Aplysia sensory neurons.

In neural mechanisms of animal learning, intracellular cAMP has been known to play an important role. In the present experiments we attempted functional transplant of a photoactivated adenylyl cyclase (PAC) isolated from Euglena into Aplysia neurons, and explored whether PAC can produce cAMP in the neurons by light stimulation. Serotonergic modulation of mechanoafferent sensory neurons in Aplysia pleural ganglia has been reported to increase intracellular cAMP level and promotes synaptic transmission to motor neurons by increasing spike width of sensory neurons. When cAMP was directly injected into the sensory neurons, spike amplitude temporarily decreased while spike width temporarily increased. This effect was not substituted by injection of 5'AMP, and maintained longer in a bath solution containing IBMX, the phosphodiesterase inhibitor. We, therefore, explored these changes as indicators of appearance of the PAC function. PAC or the PAC expression vector (pNEX-PAC) was injected into cell bodies of sensory neurons. Spike amplitude decreased in both cases and spike width increased in the PAC injection when the neurons were stimulated with light, suggesting that the transplanted PAC works well in Aplysia neurons. These results indicate that we can control cAMP production in specific neurons with light by the functional transplant of PAC.

Fluorescence behavior of Euglena photoreceptor.

To provide new information on the series of structural changes that Euglena photoreceptive proteins undergo inside the photoreceptor in response to light, we measured in vivo emission fluorescence spectra in the stable intermediates of its photocycle. Our emission spectra give a certain indication that fluorescent proteins are present in the Euglena photoreceptor and that they undergo a photocycle. On the basis of our data, we suggested that at least two stable intermediates, one of which is fluorescent, can be discriminated at room temperature and with our time resolution.