Antioxidant and Antimicrobial Potential of the Bifurcaria bifurcata Epiphytic Bacteria

Surface-associated marine bacteria are an interesting source of new secondary metabolites. The aim of this study was the isolation and identification of epiphytic bacteria from the marine brown alga, Bifurcaria bifurcata, and the evaluation of the antioxidant and antimicrobial activity of bacteria extracts. The identification of epiphytic bacteria was determined by 16S rRNA gene sequencing. Bacteria extracts were obtained with methanol and dichloromethane (1:1) extraction. The antioxidant activity of extracts was performed by quantification of total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Antimicrobial activities were evaluated against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae and Candida albicans. A total of 39 Bifurcaria bifurcata-associated bacteria were isolated and 33 were identified as Vibrio sp. (48.72%), Alteromonas sp. (12.82%), Shewanella sp. (12.26%), Serratia sp. (2.56%), Citricoccus sp. (2.56%), Cellulophaga sp. (2.56%), Ruegeria sp. (2.56%) and Staphylococcus sp. (2.56%). Six (15.38%) of the 39 bacteria Bifurcaria bifurcata-associated bacteria presented less than a 90% Basic Local Alignment Search Tool (BLAST) match, and some of those could be new. The highest antioxidant activity and antimicrobial activity (against B. subtilis) was exhibited by strain 16 (Shewanella sp.). Several strains also presented high antimicrobial activity against S. aureus, mainly belonging to Alteromonas sp. and Vibrio sp. There were no positive results against fungi and Gram-negative bacteria. Bifurcaria bifurcata epiphytic bacteria were revealed to be excellent sources of natural antioxidant and antimicrobial compounds.     

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.     

Sesquiterpene and Acetogenin Derivatives from the Marine Red Alga Laurencia okamurai

In addition to 13 known compounds, four new bisabolane sesquiterpenes, okamurenes A–D (1–4), a new chamigrane derivative, okamurene E (5), and a new C₁₂-acetogenin, okamuragenin (6), were isolated from the marine red alga Laurencia okamurai. The structures of these compounds were determined through detailed spectroscopic analyses. Of these, okamurenes A and B (1 and 2) are the first examples of bromobisabolane sesquiterpenes possessing a phenyl moiety among Laurencia-derived sesquiterpenes, while okamuragenin (6) was the first acetogenin aldehyde possessing a C₁₂-carbon skeleton. Each of the isolated compounds was evaluated for the brine shrimp (Artemia salina) lethal assay and 7-hydroxylaurene displayed potent lethality with LD₅₀ 1.8 μM.     

Pseudopterosin Biosynthesis: Aromatization of the Diterpene Cyclase Product,Elisabethatriene

Putative precursors in pseudopterosin biosynthesis, the hydrocarbons isoelisabethatriene (10) and erogorgiaene (11), have been identified from an extract of Pseudopterogorgia elisabethae collected in the Florida Keys. Biosynthetic experiments designed to test the utilization of these compounds in pseudopterosin production revealed that erogorgiaene is transformed to pseudopterosins A–D. Together with our previous data, it is now apparent that early steps in pseudopterosin biosynthesis involve the cyclization of geranylgeranyl diphosphate to elisabethatriene followed by the dehydrogenation and aromatization to erogorgiaene.     

Antibiofilm Activity of the Brown Alga Halidrys siliquosa against Clinically Relevant Human Pathogens

The marine brown alga Halidrys siliquosa is known to produce compounds with antifouling activity against several marine bacteria. The aim of this study was to evaluate the antimicrobial and antibiofilm activity of organic extracts obtained from the marine brown alga H. siliquosa against a focused panel of clinically relevant human pathogens commonly associated with biofilm-related infections. The partially fractionated methanolic extract obtained from H. siliquosa collected along the shores of Co. Donegal; Ireland; displayed antimicrobial activity against bacteria of the genus Staphylococcus; Streptococcus; Enterococcus; Pseudomonas; Stenotrophomonas; and Chromobacterium with MIC and MBC values ranging from 0.0391 to 5 mg/mL. Biofilms of S. aureus MRSA were found to be susceptible to the algal methanolic extract with MBEC values ranging from 1.25 mg/mL to 5 mg/mL respectively. Confocal laser scanning microscopy using LIVE/DEAD staining confirmed the antimicrobial nature of the antibiofilm activity observed using the MBEC assay. A bioassay-guided fractionation method was developed yielding 10 active fractions from which to perform purification and structural elucidation of clinically-relevant antibiofilm compounds.     

Antiplatelet and Anticoagulant Effects of Diterpenes Isolated from the Marine Alga,Dictyota menstrualis

Cardiovascular diseases represent a major cause of disability and death worldwide. Therapeutics are available, but they often have unsatisfactory results and may produce side effects. Alternative treatments based on the use of natural products have been extensively investigated, because of their low toxicity and side effects. Marine organisms are prime candidates for such products, as they are sources of numerous and complex substances with ecological and pharmacological effects. In this work, we investigated, through in vitro experiments, the effects of three diterpenes (pachydictyol A, isopachydictyol A and dichotomanol) from the Brazilian marine alga, Dictyota menstrualis, on platelet aggregation and plasma coagulation. Results showed that dichotomanol inhibited ADP- or collagen-induced aggregation of platelet-rich plasma (PRP), but failed to inhibit washed platelets (WP). In contrast, pachydictyol A and isopachydictyol A failed to inhibit the aggregation of PRP, but inhibited WP aggregation induced by collagen or thrombin. These diterpenes also inhibited coagulation analyzed by the prothrombin time and activated partial thromboplastin time and on commercial fibrinogen. Moreover, diterpenes inhibited the catalytic activity of thrombin. Theoretical studies using the Osiris Property Explorer software showed that diterpenes have low theoretical toxicity profiles and a drug-score similar to commercial anticoagulant drugs. In conclusion, these diterpenes are promising candidates for use in anticoagulant therapy, and this study also highlights the biotechnological potential of oceans and the importance of bioprospecting to develop medicines.     

Extracts from Cladiella australis, Clavularia viridis and Klyxum simplex (Soft Corals) are Capable of Inhibiting the Growth of Human Oral Squamous Cell Carcinoma Cells

Many biomedical products have already been obtained from marine organisms. In order to search more therapeutic drugs against cancer, this study demonstrates the cytotoxicity effects of Cladiella australis, Clavularia viridis and Klyxum simplex extracts on human oral squamous cell carcinoma (SCC4, SCC9 and SCC25) cells using cell adhesion and cell viability assay. The morphological alterations in SCCs cells after treatment with three extracts, such as typical nuclear condensation, nuclear fragmentation and apoptotic bodies of cells were demonstrated by Hoechst stain. Flow cytometry indicated that three extracts sensitized SCC25 cells in the G₀/G₁ and S-G₂/M phases with a concomitant significantly increased sub-G₁ fraction, indicating cell death by apoptosis. This apoptosis process was accompanied by activation of caspase-3 expression after SCC25 cells were treated with three extracts. Thereby, it is possible that extracts of C. australis, C. viridis and K. simplex cause apoptosis of SCCs and warrant further research investigating the possible anti-oral cancer compounds in these soft corals.     

Hydrolysis of Fucoidan by Fucoidanase Isolated from the Marine Bacterium,Formosa algae

Intracellular fucoidanase was isolated from the marine bacterium, Formosa algae strain KMM 3553. The first appearance of fucoidan enzymatic hydrolysis products in a cell-free extract was detected after 4 h of bacterial growth, and maximal fucoidanase activity was observed after 12 h of growth. The fucoidanase displayed maximal activity in a wide range of pH values, from 6.5 to 9.1. The presence of Mg²⁺, Ca²⁺ and Ba²⁺ cations strongly activated the enzyme; however, Cu²⁺ and Zn²⁺ cations had inhibitory effects on the enzymatic activity. The enzymatic activity of fucoidanase was considerably reduced after prolonged (about 60 min) incubation of the enzyme solution at 45 °C. The fucoidanase catalyzed the hydrolysis of fucoidans from Fucus evanescens and Fucus vesiculosus, but not from Saccharina cichorioides. The fucoidanase also did not hydrolyze carrageenan. Desulfated fucoidan from F. evanescens was hydrolysed very weakly in contrast to deacetylated fucoidan, which was hydrolysed more actively compared to the native fucoidan from F. evanescens. Analysis of the structure of the enzymatic products showed that the marine bacteria, F. algae, synthesized an α-l-fucanase with an endo-type action that is specific for 1→4-bonds in a polysaccharide molecule built up of alternating three- and four-linked α-l-fucopyranose residues sulfated mainly at position 2.     

Solwaric Acids A and B,Antibacterial Aromatic Acids from a Marine Solwaraspora sp.

Two novel trialkyl-substituted aromatic acids, solwaric acids A and B, were isolated from a marine Solwaraspora sp. cultivated from the ascidian Trididemnum orbiculatum. Solwaric acids A and B were isotopically labeled with U-¹³C glucose, and analysis of a ¹³C–¹³C COSY allowed for unambiguous determination of the location of the phenyl methyl group. The two novel compounds demonstrated antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA).     

Application of HPLC-NMR in the Identification of Plocamenone and Isoplocamenone from the Marine Red Alga Plocamium angustum

A combination of on-line HPLC-NMR and off-line chemical investigations has resulted in the identification of the previously reported polyhalogenated monoterpene plocamenone, together with the new structural analogue isoplocamenone from the crude extract of the marine alga Plocamium angustum. On-flow and stop-flow HPLC-NMR analyses (including the acquisition of WET 2D NMR spectra) rapidly assisted in the identification of the major component plocamenone and in the partial identification of its unstable double bond isomer isoplocamenone. Conventional off-line isolation and structural characterization techniques were employed to unequivocally confirm both structures, leading to a structural revision for plocamenone, as well as to obtain sufficient quantities for biological testing.     

Identification of Four New agr Quorum Sensing-Interfering Cyclodepsipeptides from a Marine Photobacterium

During our search for new natural products from the marine environment, we discovered a wide range of cyclic peptides from a marine Photobacterium, closely related to P. halotolerans. The chemical fingerprint of the bacterium showed primarily non-ribosomal peptide synthetase (NRPS)-like compounds, including the known pyrrothine antibiotic holomycin and a wide range of peptides, from diketopiperazines to cyclodepsipeptides of 500–900 Da. Purification of components from the pellet fraction led to the isolation and structure elucidation of four new cyclodepsipeptides, ngercheumicin F, G, H, and I. The ngercheumicins interfered with expression of virulence genes known to be controlled by the agr quorum sensing system of Staphylococcus aureus, although to a lesser extent than the previously described solonamides from the same strain of Photobacterium.     

Metabolomic Profiling and Genomic Study of a Marine Sponge-Associated Streptomyces sp

Metabolomics and genomics are two complementary platforms for analyzing an organism as they provide information on the phenotype and genotype, respectively. These two techniques were applied in the dereplication and identification of bioactive compounds from a Streptomyces sp. (SM8) isolated from the sponge Haliclona simulans from Irish waters. Streptomyces strain SM8 extracts showed antibacterial and antifungal activity. NMR analysis of the active fractions proved that hydroxylated saturated fatty acids were the major components present in the antibacterial fractions. Antimycin compounds were initially putatively identified in the antifungal fractions using LC-Orbitrap. Their presence was later confirmed by comparison to a standard. Genomic analysis of Streptomyces sp. SM8 revealed the presence of multiple secondary metabolism gene clusters, including a gene cluster for the biosynthesis of the antifungal antimycin family of compounds. The antimycin gene cluster of Streptomyces sp. SM8 was inactivated by disruption of the antimycin biosynthesis gene antC. Extracts from this mutant strain showed loss of antimycin production and significantly less antifungal activity than the wild-type strain. Three butenolides, 4,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olide (2), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (3) that had previously been reported from marine Streptomyces species were also isolated from SM8. Comparison of the extracts of Streptomyces strain SM8 and its host sponge, H. simulans, using LC-Orbitrap revealed the presence of metabolites common to both extracts, providing direct evidence linking sponge metabolites to a specific microbial symbiont.     

In Vitro Effect of the Synthetic cal14.1a Conotoxin,Derived from Conus californicus,on the Human Parasite Toxoplasma gondii

Toxins that are secreted by cone snails are small peptides that are used to treat several diseases. However, their effects on parasites with human and veterinary significance are unknown. Toxoplasma gondii is an opportunistic parasite that affects approximately 30% of the world’s population and can be lethal in immunologically compromised individuals. The conventional treatment for this parasitic infection has remained the same since the 1950s, and its efficacy is limited to the acute phase of infection. These findings have necessitated the search for new drugs that specifically target T. gondii. We examined the effects of the synthetic toxin cal14.1a (s-cal14.1a) from C. californicus on the tachyzoite form of T. gondii. Our results indicate that, at micromolar concentrations, s-cal14.1a lowers viability and inhibits host cell invasion (by 50% and 61%, respectively) on exposure to extracellular parasites. Further, intracellular replication decreased significantly while viability of the host cell was unaffected. Our study is the first report on the antiparasitic activity of a synthetic toxin of C. californicus.     

Investigation of Indolglyoxamide and Indolacetamide Analogues of Polyamines as Antimalarial and Antitrypanosomal Agents

Pure compound screening has previously identified the indolglyoxylamidospermidine ascidian metabolites didemnidine A and B (2 and 3) to be weak growth inhibitors of Trypanosoma brucei rhodesiense (IC₅₀ 59 and 44 μM, respectively) and Plasmodium falciparum (K1 dual drug resistant strain) (IC₅₀ 41 and 15 μM, respectively), but lacking in selectivity (L6 rat myoblast, IC₅₀ 24 μM and 25 μM, respectively). To expand the structure–activity relationship of this compound class towards both parasites, we have prepared and biologically tested a library of analogues that includes indoleglyoxyl and indoleacetic “capping acids”, and polyamines including spermine (PA3-4-3) and extended analogues PA3-8-3 and PA3-12-3. 7-Methoxy substituted indoleglyoxylamides were typically found to exhibit the most potent antimalarial activity (IC₅₀ 10–92 nM) but with varying degrees of selectivity versus the L6 rat myoblast cell line. A 6-methoxyindolglyoxylamide analogue was the most potent growth inhibitor of T. brucei (IC₅₀ 0.18 μM) identified in the study: it, however, also exhibited poor selectivity (L6 IC₅₀ 6.0 μM). There was no apparent correlation between antimalarial and anti-T. brucei activity in the series. In vivo evaluation of one analogue against Plasmodium berghei was undertaken, demonstrating a modest 20.9% reduction in parasitaemia.     

Discovery and Evaluation of Thiazinoquinones as Anti-Protozoal Agents

Pure compound screening has identified the dioxothiazino-quinoline-quinone ascidian metabolite ascidiathiazone A (2) to be a moderate growth inhibitor of Trypanosoma brucei rhodesiense (IC₅₀ 3.1 μM) and Plasmodium falciparum (K1 dual drug resistant strain) (IC₅₀ 3.3 μM) while exhibiting low levels of cytotoxicity (L6, IC₅₀ 167 μM). A series of C-7 amide and Δ²⁽³⁾ analogues were prepared that explored the influence of lipophilicity and oxidation state on observed anti-protozoal activity and selectivity. Little variation in anti-malarial potency was observed (IC₅₀ 0.62–6.5 μM), and no correlation was apparent between anti-malarial and anti-T. brucei activity. Phenethylamide 7e and Δ²⁽³⁾-glycine analogue 8k exhibited similar anti-Pf activity to 2 but with slightly enhanced selectivity (SI 72 and 93, respectively), while Δ²⁽³⁾-phenethylamide 8e (IC₅₀ 0.67 μM, SI 78) exhibited improved potency and selectivity towards T. brucei rhodesiense compared to the natural product hit. A second series of analogues were prepared that replaced the quinoline ring of 2 with benzofuran or benzothiophene moieties. While esters 10a/10b and 15 were once again found to exhibit cytotoxicity, carboxylic acid analogues exhibited potent anti-Pf activity (IC₅₀ 0.34–0.035 μM) combined with excellent selectivity (SI 560–4000). In vivo evaluation of a furan carboxylic acid analogue against P. berghei was undertaken, demonstrating 85.7% and 47% reductions in parasitaemia with ip or oral dosing respectively.     

Chemical Profiling (HPLC-NMR & HPLC-MS), Isolation,and Identification of Bioactive Meroditerpenoids from the Southern Australian Marine Brown Alga Sargassum paradoxum

A phytochemical investigation of a southern Australian marine brown alga, Sargassum paradoxum, resulted in the isolation and identification of four new (5, 9, 10, and 15) and nine previously reported (1, 2, 6–8, and 11–14) bioactive meroditerpenoids. HPLC-NMR and HPLC-MS were central to the identification of a new unstable compound, sargahydroquinal (9), and pivotal in the deconvolution of eight (1, 2, 5–7, and 10–12) other meroditerpenoids. In particular, the complete characterization and identification of the two main constituents (1 and 2) in the crude dichloromethane extract was achieved using stop-flow HPLC-NMR and HPLC-MS. This study resulted in the first acquisition of gHMBCAD NMR spectra in the stop-flow HPLC-NMR mode for a system solely equipped with a 60 μL HPLC-NMR flow cell without the use of a cold probe, microcoil, or any pre-concentration.     

Pseudoalteromone B: A Novel 15C Compound from a Marine Bacterium Pseudoalteromonas sp. CGH2XX

A novel 15C compound, pseudoalteromone B (1), possessing a novel carbon skeleton, was obtained from a marine bacterium Pseudoalteromonas sp. CGH2XX. This bacterium was originally isolated from a cultured-type octocoral Lobophytum crassum, that was growing in cultivating tanks equipped with a flow-through sea water system. The structure of 1 was established by spectroscopic methods. Pseudoalteromone B (1) displayed a modestly inhibitory effect on the release of elastase by human neutrophils.     

Dereplication Strategies for Targeted Isolation of New Antitrypanosomal Actinosporins A and B from a Marine Sponge Associated-Actinokineospora sp. EG49

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC₅₀ value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness.     

Drimane Sesquiterpene-Conjugated Amino Acids from a Marine Isolate of the Fungus Talaromyces minioluteus (Penicillium Minioluteum)

Four new sesquiterpene lactones (3, 4, 6 and 7) and three known compounds, purpuride (1), berkedrimane B (2) and purpuride B (5), were isolated from the marine fungus, Talaromyces minioluteus (Penicillium minioluteum). New compounds were drimane sesquiterpenes conjugated with N-acetyl-l-valine, and their structures were elucidated by analysis of spectroscopic data, as well as by single crystal X-ray analysis. The isolated compounds could not inhibit the apoptosis-regulating enzyme, caspase-3, while three of the compounds (2, 3 and 7) exhibited weak cytotoxic activity.