TSH fraction from Petromica citrina: A potential marine natural product for the treatment of sepsis by Methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND AND PURPOSE: Antibiotic resistance is now a worldwide public health problem. A potential alternative source in the search for new antibiotics is the bioactive molecules obtained from marine products, as the halistanol trisulfate, obtained from Petromica citrina, and herein investigated from its antimicrobial and anti-inflammatory properties. EXPERIMENTAL APPROACH: The antimicrobial activity of the fractionation products (TSH fraction, halistanol sulfate (HS) and halistanol sulfate C (HS-C)) of the marine sponge Petromica citrina was evaluated against twenty bacteria and two fungi strains by the disk diffusion and microdilution methods. After initial in vitro tests, an in vivo assay was proposed, to evaluate survival and inflammatory parameters in an animal model of peritonitis mediated by MRSA. The animals are treated with TSH fraction (1, 2.5 and 5 mg kg-1) or Vancomycin (30 mg kg-1) twice (6 and 18 h after induction) until organ removal for evaluation of the inflammatory profile, or for 3 days, 12 h each (6 h, 18 h, 30 h, 42 h, 54 h and 66 h after induction) in animals which were followed-up for by five days, for the evaluation of survival. KEY RESULTS: The BF fraction, TSH fraction, HS and HS-Cinhibited, in vitro, the Enterococcus faecalis, Staphylococcus aureus and Candida albicans growth. Moreover, these samples were effective against S. aureus (MSSA), MRSA and Vancomycin-Resistence Enterococcus (VRE). The in vivo results demonstrated that TSH fraction reduced mortality when compared to the saline group. To evaluate the role of inflammation in outcomes of peritonitis, cytokines (IL-1ß, IL-6, TNF-α) and MPO activity were measured. In general, anti-inflammatory activity was detected in animals treated with TSH in different doses. CONCLUSION AND IMPLICATIONS: These data suggest that TSH may be an interesting alternative for the treatment infections by Gram-positive resistant bacteria, due to its antimicrobial profile associated with its anti-inflammatory properties.

Trypanocidal activity of organic extracts from the Brazilian and Spanish marine sponges

Abstract Chagas' disease is a parasitic infection caused by protozoan Trypanosoma cruzi that affect millions of people worldwide. The available drugs for treatment of this infection cause serious side effects and have variable efficacy, especially in the chronic phase of the disease. In this context, natural compounds have shown great potential for the discovery of new chemotherapies for the treatment of this infection and various other diseases. In present study, we evaluated the in vitro antiprotozoal activity of five species of Brazilian and Spanish marine sponges (Condrosia reniformes, Tethya rubra, Tethya ignis, Mycale angulosa and Dysidea avara) against T. cruzi. By GC–MS data, we observed that in these extracts were present the major classes of the following compounds: hydrocarbons, terpenes, steroids and alcohols. The extracts showed activity against the three forms of this parasite and did not induce toxicity in mammalian cells. Better activities were observed with the extracts of marine sponges, C. reniformes (EC50 = 0.6 μg/ml), D. avara (EC50 = 1.1 μg/ml) and M. angulosa (EC50 = 3.8 μg/ml), against trypomastigote forms. In intracellular amastigote forms, the extract of T. ignis showed IC50 of 7.2 μg/ml and SI of 24.65. On this basis, our results indicate that these extracts can be promising chemotherapeutic agents against T. cruzi.

Antimicrobial (including antimollicutes), antioxidant and anticholinesterase activities of Brazilian and Spanish marine organisms – evaluation of extracts and pure compounds

Abstract This work describes the antimicrobial, antioxidant and anticholinesterase activities in vitro of organic extracts from fourteen seaweeds, eleven sponges, two ascidians, one bryozoan, and one sea anemone species collected along the Brazilian and Spanish coast, as well as the isolation of the diterpene (4R, 9S, 14S)-4α-acetoxy-9β,14α-dihydroxydolast-1(15),7-diene (1) and halogenated sesquiterpene elatol (2). The most promising antimicrobial results for cell wall bacteria were obtained by extracts from seaweeds Laurencia dendroidea and Sargassum vulgare var. nanun (MIC 250 μg/ml), and by the bryozoan Bugula neritina (MIC 62.5 μg/ml), both against Staphylococcus aureus. As for antimollicutes, extracts from seaweeds showed results better than the extracts from invertebrates. Almost all seaweeds assayed (92%) exhibited some antimicrobial activity against mollicutes strains (Mycoplasma hominis,Mycoplasma genitalium,Mycoplasma capricolum and Mycoplasma pneumoniae strain FH). From these seaweeds, A1 (Canistrocarpus cervicornis), A11 (Gracilaria sp.) and A4 (Lobophora variegata) showed the best results for M. pneumoniae strain FH (MIC 250 μg/ml). Furthermore, compounds 1 and 2 were also assayed against mollicutes strains M. hominis,M. genitalium,M. capricolum,M. pneumoniae strain 129 and M. pneumoniae strain FH, which showed MIC > 100 μg/ml. Antioxidant activities of extracts from these marine organisms were inactive, except for E7 (from sponge Ircinia sp.), which exhibited moderated antioxidant activities for two methods assayed (IC50 83.0 ± 0.1 μg/ml, and 52.0 ± 0.8 mg AA/g, respectively). Finally, for the anticholinesterase activity, all the 29 samples evaluated (100%) exhibited some level of activity, with IC50 < 1000 μg/ml. From these, seaweeds extracts were considered more promising than marine invertebrate extracts [A10 (IC50 14.4 ± 0.1 μg/ml), A16 (IC50 16.4 ± 0.4 μg/ml) and A8 (IC50 14.9 ± 0.5 μg/ml)]. The findings of this work are useful for further research aiming at isolation and characterization of active compounds.

The in vitro biological activity of the Brazilian brown seaweed Dictyota mertensii against Leishmania amazonensis.

Seaweeds present a wide variety of interesting bioactive molecules. In the present work we evaluated the biological activity of the dichloromethane/methanol (2:1) extract (DME) from the brown seaweed Dictyota mertensii against Leishmania amazonensis and its cytotoxic potential on mammalian cells. The extract showed significant inhibitory effect on the growth of promastigote forms (IC50=71.60 µg/mL) and low toxicity against mammalian cells (CC50=233.10 µg/mL). The DME was also efficient in inhibiting the infection in macrophages, with CC50 of 81.4 µg/mL and significantly decreased the survival of amastigote forms within these cells. The selectivity index showed that DME was more toxic to both promastigote (SI=3.25) and amastigote (SI=2.86) forms than to macrophages. Increased NO production was observed in treated macrophages suggesting that besides acting directly on the parasites, the DME also shows an immunomodulatory effect on macrophages. Drastic ultrastructural alterations consistent with loss of viability and cell death were observed in treated parasites. Confocal microscopy and cytometry analyzes showed no significant impairment of plasma membrane integrity, whereas an intense depolarization of mitochondrial membrane could be observed by using propidium iodide and rhodamine 123 staining, respectively. The low toxicity to mammalian cells and the effective activity against promastigotes and amastigotes, point to the use of DME as a promising agent for the treatment of cutaneous leishmaniasis.

Anti HSV-1 activity of halistanol sulfate and halistanol sulfate C isolated from Brazilian marine sponge Petromica citrina (Demospongiae).

The n-butanol fraction (BF) obtained from the crude extract of the marine sponge Petromica citrina, the halistanol-enriched fraction (TSH fraction), and the isolated compounds halistanol sulfate (1) and halistanol sulfate C (2), were evaluated for their inhibitory effects on the replication of the Herpes Simplex Virus type 1 (HSV-1, KOS strain) by the viral plaque number reduction assay. The TSH fraction was the most effective against HSV-1 replication (SI = 15.33), whereas compounds 1 (SI = 2.46) and 2 (SI = 1.95) were less active. The most active fraction and these compounds were also assayed to determine the viral multiplication step(s) upon which they act as well as their potential synergistic effects. The anti-HSV-1 activity detected was mediated by the inhibition of virus attachment and by the penetration into Vero cells, the virucidal effect on virus particles, and by the impairment in levels of ICP27 and gD proteins of HSV-1. In summary, these results suggest that the anti-HSV-1 activity of TSH fraction detected is possibly related to the synergic effects of compounds 1 and 2.

Anti-infective potential of marine invertebrates and seaweeds from the Brazilian coast.

This manuscript describes the evaluation of anti-infective potential in vitro of organic extracts from nine sponges, one ascidian, two octocorals, one bryozoan, and 27 seaweed species collected along the Brazilian coast. Antimicrobial activity was tested against Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231) by the disk diffusion method. Antiprotozoal activity was evaluated against Leishmania braziliensis (MHOM/BR/96/LSC96-H3) promastigotes and Trypanosoma cruzi (MHOM/BR/00/Y) epimastigotes by MTT assay. Activity against intracellular amastigotes of T. cruzi and L. brasiliensis in murine macrophages was also evaluated. Antiviral activity was tested against Herpes Simplex Virus type 1 (HSV-1, KOS strain) by the plaque number reduction assay (IC50). Cytotoxicity on VERO cells was evaluated by the MTT assay (CC50). The results were expressed as SI = CC50/IC50. The most promising antimicrobial results were obtained against S. aureus and C. albicans with Dragmacidon reticulatum. Among the seaweeds, only Osmundaria obtusiloba showed moderate activity against P. aeruginosa. Concerning antiprotozoal activity, Bugula neritina, Carijoa riseii, Dragmaxia anomala and Haliclona (Halichoclona) sp. showed the most interesting results, mainly against extracellular promastigote forms of L. braziliensis (66, 35.9, 97.2, and 43.6% inhibition, respectively). Moreover, six species of seaweeds Anadyomene saldanhae, Caulerpa cupressoides, Canistrocarpus cervicornis, Dictyota sp., Ochtodes secundiramea, and Padina sp. showed promising results against L. braziliensis (87.9, 51.7, 85.9, 93.3, 99.7, and 80.9% inhibition, respectively), and only Dictyota sp. was effective against T. cruzi (60.4% inhibition). Finally, the antiherpes activity was also evaluated, with Haliclona (Halichoclona) sp. and Petromica citrina showing the best results (SI = 11.9 and SI > 5, respectively). All the active extracts deserve special attention in further studies to chemically characterize the bioactive compounds, and to perform more refined biological assays.

Screening of Dengue virus antiviral activity of marine seaweeds by an in situ enzyme-linked immunosorbent assay.

Dengue is a significant public health problem worldwide. Despite the important social and clinical impact, there is no vaccine or specific antiviral therapy for prevention and treatment of dengue virus (DENV) infection. Considering the above, drug discovery research for dengue is of utmost importance; in addition natural marine products provide diverse and novel chemical structures with potent biological activities that must be evaluated. In this study we propose a target-free approach for dengue drug discovery based on a novel, rapid, and economic in situ enzyme-linked immunosorbent assay and the screening of a panel of marine seaweed extracts. The in situ ELISA was standardized and validated for Huh7.5 cell line infected with all four serotypes of DENV, among them clinical isolates and a laboratory strain. Statistical analysis showed an average S/B of 7.2 and Z-factor of 0.62, demonstrating assay consistency and reliability. A panel of fifteen seaweed extracts was then screened at the maximum non-toxic dose previously determined by the MTT and Neutral Red cytotoxic assays. Eight seaweed extracts were able to reduce DENV infection of at least one serotype tested. Four extracts (Phaeophyta: Canistrocarpus cervicornis, Padina gymnospora; Rhodophyta: Palisada perforate; Chlorophyta: Caulerpa racemosa) were chosen for further evaluation, and time of addition studies point that they might act at an early stage of the viral infection cycle, such as binding or internalization.

Acaricidal and repellent activity of terpenoids from seaweeds Collected in Pernambuco, Brazil.

n-Hexane and dichloromethane extracts obtained from Laurencia dentroidea (Rhodophyta) and Canistrocarpus cervicornis (Phaeophyta) were investigated for their acaricidal and repellent properties against Tetranychus urticae under laboratory conditions. The two extracts displayed moderate toxicity and good repellent proprieties, and were significantly more toxic (36-fold) than the positive control (eugenol), whereas eugenol was tenfold more repellent than either seaweed extract. The sesquiterpenoid elatol (1) was isolated from L. dentroidea and the diterpenoid seco-dolastane (4R,9S,14S)-4alpha-acetoxy-9beta, 14alpha-dihydroxydolast-1(15),7-diene (2) from C. cervicornis, the chemical structures of which were characterized by NMR spectroscopic data (1H and 13C) and by comparison with literature data. These compounds exhibited moderate toxicity, but a high degree of repellent activity against T. urticae. The findings suggest that marine natural products, specifically terpenes, can be employed for the development of new pesticides and become prototype agrochemical agents.

Oviposition and Embryotoxicity of Indigofera suffruticosa on Early Development of Aedes aegypti (Diptera: Culicidae).

Aqueous extract of Indigofera suffruticosa leaves obtained by infusion was used to evaluate the oviposition, its effect on development of eggs and larvae, and morphological changes in larvae of Aedes aegypti. The bioassays were carried out with aqueous extract in different concentrations on eggs, larvae, and female mosquitoes, and the morphological changes were observed in midgut of larvae. The extract showed repellent activity on A. aegypti mosquitoes, reducing significantly the egg laying by females with control substrate (343 (185-406)) compared with the treated substrate (88 (13-210)). No eclosion of A. aegypti eggs at different concentrations studied was observed. The controleclodedin 35%. At concentration of 250 µg/mL, 93.3% of larvae remained in the second instar of development and at concentrations of 500, 750, and 1000 µg/mL the inhibitory effect was lower with percentages of 20%, 53.3%, and 46.6%, respectively. Morphological changes like disruption on the peritrophic envelope (PE), discontinued underlying epithelium, increased gut lumen, and segments with hypertrophic aspects were observed in anterior region of medium midgut of larvae of A. aegypti. The results showed repellent activity, specific embryotoxicity, and general growth retardation in A. aegypti by medium containing aqueous extract of I. suffruticosa leaves.

4-Acetoxydolastane diterpene from the Brazilian brown alga Canistrocarpus cervicornis as antileishmanial agent.

Natural marine products have shown an interesting array of diverse and novel chemical structures with potent biological activities. Our study reports the antiproliferative assays of crude extracts, fraction and pure compound (4R,9S,14S)-4α-acetoxy-9ß,14α-dihydroxydolast-1(15),7-diene (1) obtained from brown alga Canistrocarpus cervicornis showing the antileishmanial activity. We showed that 1 had a dose-dependent activity during 72 h of treatment, exhibiting IC(50) of 2.0 µg/mL, 12.0 µg/mL, and 4.0 µg/mL for promastigote, axenic amastigote and intracellular amastigote forms of Leishmania amazonensis, respectively. A cytotoxicity assay showed that the action of the isolated compound 1 was 93.0 times less toxic to the macrophage than to the protozoan. Additionally, compound 1 induced ultrastructural changes, including extensive mitochondrial damage; decrease in Rh123 fluorescence, suggesting interference with the mitochondrial membrane potential; and lipid peroxidation in parasite cells. The use of 1 from C. cervicornis against L. amazonensis parasites might be of great interest as a future alternative to the development of new antileishmanial drugs.

Anticoagulation and antiplatelet effects of a dolastane diterpene isolated from the marine brown alga Canistrocarpus cervicornis.

Marine brown algae of the family Dictyotaceae are rich sources of monocyclic, bicyclic, and tricyclic diterpenes. These molecules are responsible for a wide range of pharmacological and ecological functions, as antitumor and antiviral. Here, we analyzed the effect of the dolastane diterpene (4R, 9S, 14S)-4α-Acetoxy-9ß,14α-dihydroxydolast-1(15),7-diene, isolated from the marine brown alga, Canistrocarpus cervicornis on blood clotting and platelet aggregation. The dolastane diterpene was able to inhibit either plasma or fibrinogen coagulation induced by thrombin as well as delayed coagulation in the recalcification test. The dolastane diterpene impaired, in a concentration-dependent manner platelet aggregation induced by collagen or adenosine diphosphate with no lysis on such cells. Thus, the dolastane diterpene maybe a promising source of natural inhibitors for hemostatic disturbs (clotting and platelet aggregation) leading to the discovery of drugs of potential use as antithrombotic and antiplatelet. In addition, the dolastane diterpene may be used as a molecular model for development of new antithrombotic agents giving new approaches to the management to the treatment of thrombotic disturbs.

Effect of elatol, isolated from red seaweed Laurencia dendroidea, on Leishmania amazonensis.

In the present study, we investigated the antileishmanial activity of sesquiterpene elatol, the major constituent of the Brazilian red seaweed Laurencia dendroidea (Hudson) J.V. Lamouroux, against L. amazonensis. Elatol after 72 h of treatment, showed an IC(50) of 4.0 µM and 0.45 µM for promastigote and intracellular amastigote forms of L. amazonensis, respectively. By scanning and transmission electron microscopy, parasites treated with elatol revealed notable changes compared with control cells, including: pronounced swelling of the mitochondrion; appearance of concentric membrane structures inside the organelle; destabilization of the plasma membrane; and formation of membrane structures, apparently an extension of the endoplasmic reticulum, which is suggestive of an autophagic process. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa, and it is not toxic to macrophages. Our studies indicated that elatol is a potent antiproliferative agent against promastigote and intracellular amastigote forms, and may have important advantages for the development of new anti-leishamanial chemotherapies.

WITHDRAWN: Antiophidian properties of a dolastane diterpene isolated from the marine brown alga Canistrocarpus cervicornis.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.bionut.2011.06.021. The duplicate article has therefore been withdrawn.

Antilonomic effects of Brazilian brown seaweed extracts.

The aim of this work was to investigate the hemolysis and blood clotting activity of Lomonia obliqua venom and the ability of some Brazilian marine algal extracts (Canistrocarpus cervicornis, Stypopodium zonale and Dictyota pfaffi) to antagonize such biological activities. L. obliqua caterpillars are dangerous to human beings and envenomation symptoms are characterized by hemorrhagic, hemolytic and blood clotting disorders, and acute renal failure, which sometimes lead to the death of the victims. Through in vitro experiments we have shown that L. obliqua venom is able to clot human plasma and hemolize human erythrocytes and that the coagulation activity of the venom is inhibited by the extracts of C. cervicornis, S. zonale and D. pfaffi. In contrast, C. cervicornis and S. zonale extracts did not inhibit the hemolytic activity of L. oblqua, as did the extract of D. pfaffi. These finding indicate that marine algae may be used as antivenoms or may contribute to the development of compounds with antilonomic effects.

Brown seaweed defensive chemicals: a structure-activity relationship approach for the marine environment.

The literature describes several diterpenes from brown seaweeds that act as defensive chemicals against natural enemies, such as competitors, epiphytes, pathogenic bacteria and herbivores. A structure-activity relationship is here presented using a new molecular modeling approach to identify structural and chemical features important to the defensive profile of four structurally related diterpenes (three dolastanes and one seco-dolastane) from Canistrocarpus cervicornis against the feeding process of the omnivorous sea urchin Lytechinus variegatus. Our experimental data revealed the herbivory inhibitory profile (HIE) for three of these evaluated compounds with (4R,7R, 14S)-4alpha,7alpha-diacetoxy-14-hydroxydolast-1(15),8-diene presenting the highest effect (HIE = 70%). Interestingly, the molecular modeling results infer that this biological activity seems to be related to several different structural features, including HOMO distribution, the molecular structure conformation, and the fulfillment of minimum requirements regarding molecular weight. These results reinforce the hypothesis about the intricate biological mechanism of these molecules due to the complexity of their chemical structures. Our work may help in the understanding of these defensive mechanisms and point to a new perspective of ecological and/or evolutionary evaluation in this area.

Inhibition of mammal Na(+)K(+)-ATPase by diterpenes extracted from the Brazilian brown alga Dictyota cervicornis.

The dolastane diterpenes 4-acetoxy-9,14-dihydroxydolast-1(15),7-diene (1) and 4,7-diacetoxy-14-hydroxydolast-1(15),8-diene (2) were isolated from specimens of the alga Dictyota cervicornis collected from the Rio de Janeiro coast, Brazil. Chemical structures of the diterpenes were assigned by 1D and 2D NMR spectral data for the first time. Both substances inhibited Na(+)K(+)-ATPase preparations from guinea-pig brain or kidney, with the same inhibitory potency towards enzyme isoforms. The maximal inhibition obtained for 1 was 40% at a concentration of 0.5 mm in the incubation mixture, while it reached 80% for compound 2 at this concentration. Ouabain insensitive ATPases were inhibited by 1, but not by 2. Data comparing the inhibitory potency of these compounds with that of ouabain and oleic acid suggest a higher degree of selectivity of 2 towards the Na(+)K(+)-pump. Cardiac glycosides such as ouabain are used classically in the treatment of heart failure, but alterations of Na(+)K(+)-pump activity are also involved in several other diseases. Therefore, the study of compounds interfering with this pump activity is gaining further importance.