Antiprotozoal activities of organic extracts from French marine seaweeds

Marine macrophytes contain a variety of biologically active compounds, some reported to have antiprotozoal activity in vitro. As a part of a screening program to search for new natural antiprotozoals, we screened hydroalcoholic and ethyl acetate extracts of 20 species of seaweeds from three phyla (Rhodophyta, Heterokontophyta and Chlorophyta), sampled along the Normandy (France) coast. We tested them in vitro against the protozoa responsible for three major endemic parasitic diseases: Plasmodium falciparum, Leishmania donovani and Trypanosoma cruzi. The selectivity of the extracts was also evaluated by testing on a mammalian cell line (L6 cells). Ethyl acetate extracts were more active than hydroalcoholic ones. Activity against T. cruzi and L. donovani was non-existent to average, but almost half the extracts showed good activity against P. falciparum. The ethyl acetate extract of Mastocarpus stellatus showed the best antiplasmodial activity as well as the best selectivity index (IC(50) = 2.8 μg/mL; SI > 30). Interestingly, a red algae species, which shares phylogenetic origins with P. falciparum, showed the best antiplasmodial activity. This study is the first to report comparative antiprotozoal activity of French marine algae. Some of the species studied here have not previously been biologically evaluated.     

Aspergillus Sydowii Marine Fungal Bloom in Australian Coastal Waters,Its Metabolites and Potential Impact on Symbiodinium Dinoflagellates

Dust has been widely recognised as an important source of nutrients in the marine environment and as a vector for transporting pathogenic microorganisms. Disturbingly, in the wake of a dust storm event along the eastern Australian coast line in 2009, the Continuous Plankton Recorder collected masses of fungal spores and mycelia (~150,000 spores/m³) forming a floating raft that covered a coastal area equivalent to 25 times the surface of England. Cultured A. sydowii strains exhibited varying metabolite profiles, but all produced sydonic acid, a chemotaxonomic marker for A. sydowii. The Australian marine fungal strains share major metabolites and display comparable metabolic diversity to Australian terrestrial strains and to strains pathogenic to Caribbean coral. Secondary colonisation of the rafts by other fungi, including strains of Cladosporium, Penicillium and other Aspergillus species with distinct secondary metabolite profiles, was also encountered. Our bioassays revealed that the dust-derived marine fungal extracts and known A. sydowii metabolites such as sydowic acid, sydowinol and sydowinin A adversely affect photophysiological performance (F_v/F_m) of the coral reef dinoflagellate endosymbiont Symbiodinium. Different Symbiodinium clades exhibited varying sensitivities, mimicking sensitivity to coral bleaching phenomena. The detection of such large amounts of A. sydowii following this dust storm event has potential implications for the health of coral environments such as the Great Barrier Reef.     

Phylogeny of Penicillium and the segregation of Trichocomaceae into three families

Species of Trichocomaceae occur commonly and are important to both industry and medicine. They are associated with food spoilage and mycotoxin production and can occur in the indoor environment, causing health hazards by the formation of β-glucans, mycotoxins and surface proteins. Some species are opportunistic pathogens, while others are exploited in biotechnology for the production of enzymes, antibiotics and other products. Penicillium belongs phylogenetically to Trichocomaceae and more than 250 species are currently accepted in this genus. In this study, we investigated the relationship of Penicillium to other genera of Trichocomaceae and studied in detail the phylogeny of the genus itself. In order to study these relationships, partial RPB1, RPB2 (RNA polymerase II genes), Tsr1 (putative ribosome biogenesis protein) and Cct8 (putative chaperonin complex component TCP-1) gene sequences were obtained. The Trichocomaceae are divided in three separate families: Aspergillaceae, Thermoascaceae and Trichocomaceae. The Aspergillaceae are characterised by the formation flask-shaped or cylindrical phialides, asci produced inside cleistothecia or surrounded by Hülle cells and mainly ascospores with a furrow or slit, while the Trichocomaceae are defined by the formation of lanceolate phialides, asci borne within a tuft or layer of loose hyphae and ascospores lacking a slit. Thermoascus and Paecilomyces, both members of Thermoascaceae, also form ascospores lacking a furrow or slit, but are differentiated from Trichocomaceae by the production of asci from croziers and their thermotolerant or thermophilic nature. Phylogenetic analysis shows that Penicillium is polyphyletic. The genus is re-defined and a monophyletic genus for both anamorphs and teleomorphs is created (Penicillium sensu stricto). The genera Thysanophora, Eupenicillium, Chromocleista, Hemicarpenteles and Torulomyces belong in Penicilliums. str. and new combinations for the species belonging to these genera are proposed. Analysis of Penicillium below genus rank revealed the presence of 25 clades. A new classification system including both anamorph and teleomorph species is proposed and these 25 clades are treated here as sections. An overview of species belonging to each section is presented. TAXONOMIC NOVELTIES: New sections, all in Penicillium: sect. Sclerotiora Houbraken & Samson, sect. Charlesia Houbraken & Samson, sect. Thysanophora Houbraken & Samson,sect. Ochrosalmonea Houbraken & Samson, sect. Cinnamopurpurea Houbraken & Samson, Fracta Houbraken & Samson, sect. Stolkia Houbraken & Samson, sect. Gracilenta Houbraken & Samson, sect. Citrina Houbraken & Samson, sect. Turbata Houbraken & Samson, sect. Paradoxa Houbraken & Samson, sect. Canescentia Houbraken & Samson. New combinations:Penicillium asymmetricum (Subramanian & Sudha) Houbraken & Samson, P. bovifimosum (Tuthill & Frisvad) Houbraken & Samson, P. glaucoalbidum (Desmazières) Houbraken & Samson, P. laeve (K. Ando & Manoch) Houbraken & Samson, P. longisporum (Kendrick) Houbraken & Samson, P. malachiteum (Yaguchi & Udagawa) Houbraken & Samson, P. ovatum (K. Ando & Nawawi) Houbraken & Samson, P. parviverrucosum (K. Ando & Pitt) Houbraken & Samson, P. saturniforme (Wang & Zhuang) Houbraken & Samson, P. taiwanense (Matsushima) Houbraken & Samson. New names:Penicillium coniferophilum Houbraken & Samson, P. hennebertii Houbraken & Samson, P. melanostipe Houbraken & Samson, P. porphyreum Houbraken & Samson.     

Insights into the Toxicological Properties of a Low Molecular Weight Fraction from Zoanthus sociatus (Cnidaria)

The phylum Cnidaria is an ancient group of venomous animals, specialized in the production and delivery of toxins. Many species belonging to the class Anthozoa have been studied and their venoms often contain a group of peptides, less than 10 kDa, that act upon ion channels. These peptides and their targets interact with high affinity producing neurotoxic and cardiotoxic effects, and even death, depending on the dose and the administration pathway. Zoanthiniaria is an order of the Subclass Hexacorallia, class Anthozoa, and unlike sea anemone (order Actiniaria), neither its diversity of toxins nor the in vivo effects of the venoms has been exhaustively explored. In this study we assessed some toxicological tests on mice with a low molecular weight fraction obtained by gel filtration in Sephadex G-50 from Zoanthus sociatus crude extract. The gel filtration chromatogram at 280 nm revealed two major peaks, the highest absorbance corresponding to the low molecular weight fraction. The toxicological effects seem to be mostly autonomic and cardiotoxic, causing death in a dose dependent manner with a LD₅₀ of 792 μg/kg. Moreover, at a dose of 600 μg/kg the active fraction accelerated the KCl-induced lethality in mice.     

Inhibition of Bacterial Quorum Sensing by Extracts from Aquatic Fungi: First Report from Marine Endophytes

In our search for quorum-sensing (QS) disrupting molecules, 75 fungal isolates were recovered from reef organisms (endophytes), saline lakes and mangrove rhizosphere. Their QS inhibitory activity was evaluated in Chromobacterium violaceum CVO26. Four strains of endophytic fungi stood out for their potent activity at concentrations from 500 to 50 μg mL⁻¹. The molecular characterization, based on the internal transcribed spacer (ITS) region sequences (ITS1, 5.8S and ITS2) between the rRNA of 18S and 28S, identified these strains as belonging to four genera: Sarocladium (LAEE06), Fusarium (LAEE13), Epicoccum (LAEE14), and Khuskia (LAEE21). Interestingly, three came from coral species and two of them came from the same organism, the coral Diploria strigosa. Metabolic profiles obtained by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) suggest that a combination of fungal secondary metabolites and fatty acids could be the responsible for the observed activities. The LC-HRMS analysis also revealed the presence of potentially new secondary metabolites. This is, to the best of our knowledge, the first report of QS inhibition by marine endophytic fungi.     

Astaxanthin: a potential therapeutic agent in cardiovascular disease

Astaxanthin is a xanthophyll carotenoid present in microalgae, fungi, complex plants, seafood, flamingos and quail. It is an antioxidant with anti-inflammatory properties and as such has potential as a therapeutic agent in atherosclerotic cardiovascular disease. Synthetic forms of astaxanthin have been manufactured. The safety, bioavailability and effects of astaxanthin on oxidative stress and inflammation that have relevance to the pathophysiology of atherosclerotic cardiovascular disease, have been assessed in a small number of clinical studies. No adverse events have been reported and there is evidence of a reduction in biomarkers of oxidative stress and inflammation with astaxanthin administration. Experimental studies in several species using an ischaemia-reperfusion myocardial model demonstrated that astaxanthin protects the myocardium when administered both orally or intravenously prior to the induction of the ischaemic event. At this stage we do not know whether astaxanthin is of benefit when administered after a cardiovascular event and no clinical cardiovascular studies in humans have been completed and/or reported. Cardiovascular clinical trials are warranted based on the physicochemical and antioxidant properties, the safety profile and preliminary experimental cardiovascular studies of astaxanthin.     

Carotenoid β-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls

Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C₄₀-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′)-ketolase (4(4′)-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3′)-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′)-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2′)-hydroxylated carotenoids).     

Eleganolone,a Diterpene from the French Marine Alga Bifurcaria bifurcata Inhibits Growth of the Human Pathogens Trypanosoma brucei and Plasmodium falciparum

Organic extracts of 20 species of French seaweed have been screened against Trypanosoma brucei rhodesiense trypomastigotes, the parasite responsible for sleeping sickness. These extracts have previously shown potent antiprotozoal activities in vitro against Plasmodium falciparum and Leishmania donovani. The selectivity of the extracts was also evaluated by testing cytotoxicity on a mammalian L6 cell line. The ethyl acetate extract of the brown seaweed, Bifurcaria bifurcata, showed strong trypanocidal activity with a mild selectivity index (IC₅₀ = 0.53 µg/mL; selectivity index (SI) = 11.6). Bio-guided fractionation led to the isolation of eleganolone, the main diterpenoid isolated from this species. Eleganolone contributes only mildly to the trypanocidal activity of the ethyl acetate extract (IC₅₀ = 45.0 µM, SI = 4.0). However, a selective activity against P. falciparum erythrocytic stages in vitro has been highlighted (IC₅₀ = 7.9 µM, SI = 21.6).     

Sarcocrassocolides M-O, bioactive cembranoids from the Dongsha Atoll soft coral Sarcophyton crassocaule

Three new cembranoids, sarcocrassocolides M-O (1-3), have been isolated from the soft coral Sarcophyton crassocaule. The structures of the metabolites were determined by extensive spectroscopic analysis. Compounds 1-3 were shown to exhibit moderate cytotoxicity toward a limited panel of cancer cell lines and display significant in vitro anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells by inhibiting the expression of the iNOS protein.     

Astaxanthin from Haematococcus pluvialis Prevents Oxidative Stress on Human Endothelial Cells without Toxicity

Astaxanthin, a powerful antioxidant, is a good candidate for the prevention of intracellular oxidative stress. The aim of the study was to compare the antioxidant activity of astaxanthin present in two natural extracts from Haematococcus pluvialis, a microalgae strain, with that of synthetic astaxanthin. Natural extracts were obtained either by solvent or supercritical extraction methods. UV, HPLC-DAD and (HPLC-(atmospheric pressure chemical ionization (APCI)+)/ion trap-MS) characterizations of both natural extracts showed similar compositions of carotenoids, but different percentages in free astaxanthin and its ester derivatives. The Trolox equivalent antioxidant capacity (TEAC) assay showed that natural extracts containing esters displayed stronger antioxidant activities than free astaxanthin. Their antioxidant capacities to inhibit intracellular oxidative stress were then evaluated on HUVEC cells. The intracellular antioxidant activity in natural extracts was approximately 90-times higher than synthetic astaxanthin (5 µM). No modification, neither in the morphology nor in the viability, of vascular human cells was observed by in vitro biocompatibility study up to 10 µM astaxanthin concentrations. Therefore, these results revealed the therapeutic potential of the natural extracts in vascular human cell protection against oxidative stress without toxicity, which could be exploited in prevention and/or treatment of cardiovascular diseases.     

In vivo and in vitro anti-inflammatory activity of neorogioltriol, a new diterpene extracted from the red algae Laurencia glandulifera

Neorogioltriol is a tricyclic brominated diterpenoid isolated from the organic extract of the red algae Laurencia glandulifera. In the present study, the anti-inflammatory effects of neorogioltriol were evaluated both in vivo using carrageenan-induced paw edema and in vitro on lipopolysaccharide (LPS)-treated Raw264.7 macrophages. The in vivo study demonstrated that the administration of 1 mg/kg of neorogioltriol resulted in the significant reduction of carregeenan-induced rat edema. In vitro, our results show that neorogioltriol treatment decreased the luciferase activity in LPS-stimulated Raw264.7 cells, stably transfected with the NF-κB-dependent luciferase reporter. This effect on NF-κB activation is not mediated through MAPK pathways. The inhibition of NF-κB activity correlates with decreased levels of LPS-induced tumor necrosis factor-alpha (TNFα) present in neorogioltriol treated supernatant cell culture. Further analyses indicated that this product also significantly inhibited the release of nitric oxide and the expression of cyclooxygenase-2 (COX-2) in LPS-stimulated Raw264.7 cells. These latter effects could only be observed for neorogioltriol concentrations below 62.5 μM. To our knowledge, this is the first report describing a molecule derived from Laurencia glandulifera with anti-inflammatory activity both in vivo and in vitro. The effect demonstrated in vitro may be explained by the inhibition of the LPS-induced NF-κB activation and TNFα production. NO release and COX-2 expression may reinforce this effect.     

Three New Cembranoids from the Taiwanese Soft Coral Sarcophyton ehrenbergi

In order to search for new bioactive substances from marine organisms, we have investigated the acetone extracts of the soft coral Sarcophyton ehrenbergi collected at San-Hsian-Tai, Taitong County, Taiwan. Chromatographic fractionation of the extracts of the octocoral S. ehrenbergi led to the isolation of three new cembranoids, (+)-12-ethoxycarbonyl-11Z-sarcophine (1), ehrenbergol A and B (2 and 3). The structures of these isolated metabolites were elucidated through extensive spectroscopic analyses. Moreover, metabolites 1-3 were evaluated in vitro for their cytotoxicity towards selected cancer cell lines and antiviral activity against human cytomegalovirus (HCMV).     

Cembranoids from the Dongsha Atoll soft coral Lobophytum crassum

Chemical investigation of the Dongsha Atoll soft coral Lobophytum crassum has afforded four new cembranoids, crassumols A-C (1-3) and 13-acetoxysarcophytoxide (4). The structures of these isolated compounds were elucidated by extensive NMR and HRESIMS experiments. The cytotoxicity and anti-HCMV (Human cytomegalovirus) activities of 1-4 were evaluated in vitro. Compound 4 exhibited cytotoxicity against A-549 (human lung carcinoma) cell line with an ED(50) of 3.6 μg/mL.     

Sulfated Galactan from Palisada flagellifera Inhibits Toxic Effects of Lachesis muta Snake Venom

In Brazil, snakebites are a public health problem and accidents caused by Lachesis muta have the highest mortality index. Envenomation by L. muta is characterized by systemic (hypotension, bleeding and renal failure) and local effects (necrosis, pain and edema). The treatment to reverse the evolution of all the toxic effects is performed by injection of antivenom. However, such therapy does not effectively neutralize tissue damage or any other local effect, since in most cases victims delay seeking appropriate medical care. In this way, alternative therapies are in demand, and molecules from natural sources have been exhaustively tested. In this paper, we analyzed the inhibitory effect of a sulfated galactan obtained from the red seaweed Palisada flagellifera against some toxic activities of L. muta venom. Incubation of sulfated galactan with venom resulted in inhibition of hemolysis, coagulation, proteolysis, edema and hemorrhage. Neutralization of hemorrhage was also observed when the galactan was administered after or before the venom injection; thus mimicking a real in vivo situation. Moreover, the galactan blocked the edema caused by a phospholipase A₂ isolated from the same venom. Therefore, the galactan from P. flagellifera may represent a promising tool to treat envenomation by L. muta as a coadjuvant for the conventional antivenom.     

Capilloquinol: a novel farnesyl quinol from the Dongsha atoll soft coral Sinularia capillosa

Capilloquinol (1), possessing an unprecedented farnesyl quinoid skeleton, was isolated from the Dongsha Atoll soft coral Sinularia capillosa. The structure of capilloquinol was elucidated by extensive analysis of spectroscopic data. The cytotoxicity and antiviral activity against human cytomegalovirus of 1 was evaluated in vitro.     

Response Surface Methodology for Ultrasound-Assisted Extraction of Astaxanthin from Haematococcus pluvialis

Astaxanthin is a novel carotenoid nutraceutical occurring in many crustaceans and red yeasts. It has exhibited various biological activities including prevention or amelioration of cardiovascular disease, gastric ulcer, hypertension, and diabetic nephropathy. In this study, ultrasound-assisted extraction was developed for the effective extraction of astaxanthin from Haematococcus pluvialis. Some parameters such as extraction solvent, liquid-to-solid ratio, extraction temperature, and extraction time were optimized by single-factor experiment and response surface methodology. The optimal extraction conditions were 48.0% ethanol in ethyl acetate, the liquid-to-solid ratio was 20:1 (mL/g), and extraction for 16.0 min at 41.1 °C under ultrasound irradiation of 200 W. Under optimal conditions, the yield of astaxanthin was 27.58 ± 0.40 mg/g. The results obtained are beneficial for the full utilization of Haematococcus pluvialis, which also indicated that ultrasound-assisted extraction is a very useful method for extracting astaxanthin from marine life.     

Effects of temperature and photoperiod on flowering time of forage legumes in a Mediterranean environment

Flowering time plasticity is a commonly occurring adaptive characteristic of fodder crops, including legumes, in arid and semiarid environments of the Mediterranean regions. Time of flowering is mainly influenced by genotype, temperature and photoperiod. Field experiments were carried out at Foggia (southern Italy) during successive growing seasons (from 8 to 16 growing cycles according to species) to study the relation among air temperature, photoperiod and duration of the morphological development of flowering in eight forage legume species: sulla (Hedysarum coronarium L.), sainfoin (Onobrychis viciifolia Scop.), pea (Pisum sativun L.), berseem clover (Trifolium alexandrinum L.), Persian clover (Trifolium resupinatum L.), faba bean (Vicia faba L.), common vetch (Vicia sativa L.) and hairy vetch (Vicia villosa Roth). Time to reach 10% flowering (EF) and 100% flowering (FF) were recorded. Rate of progress to flowering, defined as the inverse of time from sowing to EF and FF, was related to mean daily temperature, or to both mean daily temperature and mean photoperiod. Using the linear equations, the thermal time requirements (T_t) and the base temperature (T_b) expressed as heat units were determined by the x-intercept method for both EF and FF stages. Evaluation of flowering time was also based on days after planting (DAP), day of year (DOY) and on a photothermal index (PTI). For all species, a significant negative correlation (P ≥ 0.01) was found between planting date (PD) and DAP whereas PTI showed a significant negative relationship (P ≥ 0.05) only for faba bean, pea, berseem clover and common vetch. In sainfoin, sulla and berseem clover, the rate of progress to flowering was affected significantly (P ≥ 0.05) by both mean temperature and photoperiod. The T_t requirements to reach the EF and the FF stage ranged from 871 to 1665 °C day and from 1043 to 1616 °C day, respectively, for the studied species. Both phenological stages considered depended upon accumulated thermal time above a species-specific base temperature. Furthermore, in all legumes the onset of flowering only occurred when dual thresholds of a minimum T_t and a minimum photoperiod were reached, which were specific to each species.     

Bioactive Polyoxygenated Steroids from the South China Sea Soft Coral,Sarcophyton sp

Seven new polyoxygenated steroids (1–7) were isolated together with seven known analogues (8–14) from the South China Sea soft coral, Sarcophyton sp. The structures of the new compounds were identified on the basis of extensive spectroscopic analysis and comparison with reported data. All the steroids are characterized with 3β,5α,6β-hydroxy moiety, displaying carbon skeletons of cholestane, ergostane, gorgostane and 23,24-dimethyl cholestane. In the in vitro bioassay, metabolites exhibited different levels of antimicrobial activity against bacterial species Escherichia coli and Bacillus megaterium, and fungal species Microbotryum violaceum and Septoria tritici. No inhibition was detected towards microalga Chlorella fusca. Preliminary structure-activity analysis suggests that the 11α-acetoxy group may increase both antibacterial and antifungal activities. The terminal-double bond and the cyclopropane moiety at the side chain may also contribute to the bioactivity.     

Chlorella zofingiensis as an Alternative Microalgal Producer of Astaxanthin: Biology and Industrial Potential

Astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione), a high-value ketocarotenoid with a broad range of applications in food, feed, nutraceutical, and pharmaceutical industries, has been gaining great attention from science and the public in recent years. The green microalgae Haematococcus pluvialis and Chlorella zofingiensis represent the most promising producers of natural astaxanthin. Although H. pluvialis possesses the highest intracellular astaxanthin content and is now believed to be a good producer of astaxanthin, it has intrinsic shortcomings such as slow growth rate, low biomass yield, and a high light requirement. In contrast, C. zofingiensis grows fast phototrophically, heterotrophically and mixtrophically, is easy to be cultured and scaled up both indoors and outdoors, and can achieve ultrahigh cell densities. These robust biotechnological traits provide C. zofingiensis with high potential to be a better organism than H. pluvialis for mass astaxanthin production. This review aims to provide an overview of the biology and industrial potential of C. zofingiensis as an alternative astaxanthin producer. The path forward for further expansion of the astaxanthin production from C. zofingiensis with respect to both challenges and opportunities is also discussed.