Chemical diversity and antifouling activity of geniculate calcareous algae (Corallinales, Rhodophyta) from Brazil.

Marine biofouling is a natural process by which many organisms colonize and grow in submerged structures, causing serious economic consequences for the maritime industry. Geniculate calcareous algae (GCA; Corallinales, Rhodophyta) produce bioactive secondary metabolites and are a promise for new antifouling compounds. Here, we investigated the antifouling activity of four GCA species-Amphiroa beauvoisii, Jania sagittata (formerly Cheilosporum sagittatum), Jania crassa, and Jania prolifera (formerly Amphiroa flabellata)-from the Brazilian coast against macro- and microorganisms. Simultaneously, metabolomic tools were applied to assess the chemical profiles of these seaweeds using gas chromatography coupled to mass spectrometry (GC-MS). Data analysis by principal component and molecular networking analyses used the global natural products social molecular networking platform (GNPS). Our results showed that all extracts were active against different strains of marine bacteria and that the J. sagittata (JsSI) extract showed the highest percentage of bacterial inhibition. The J. sagittata (JsSI) extract was the most active against the mussel Perna perna, showing 100% byssus inhibition. Regarding toxicity, only the J. crassa (JcP) extract showed a 20% mortality rate. The chemical profiles of the evaluated GCA extracts differed qualitatively and quantitatively. Yet, the steroid (3ß)-cholest-5-en-3-ol was the major compound commonly identified in all extracts, with the exception of J. sagittata (JsSI). Moreover, we observed intra- and interspecific chemical variabilities among GCA extracts for the different populations, which could explain their antifouling activity variability. This study contributed new information about the chemical compounds produced by this group of seaweeds and showed its antifouling potential. These GCA species may be the subject of future studies to obtain new bioactive compounds with biotechnological potential in maritime areas.

Partial characterization and anticoagulant activity of sulfated galactan from the green seaweed Halimeda opuntia.

The number of deaths associated with cardiovascular diseases (CVD) increases every year, leading to an intense search for new compounds that may be employed as anticoagulants. One of the classes of bioprospected molecules comprises sulfated polysaccharides (SP) from seaweed, as heparin displays many adverse effects associated with its use. The present study aimed to characterize and evaluate the anticoagulant potential of SP extracted from the green algae Halimeda opuntia. Four PS-rich fractions, F23, F44, F60 and F75, were obtained by proteolytic digestion in papain followed by ethanol precipitation. The presence of SP was confirmed by agarose gel electrophoresis, revealing different populations in each fraction. The F44 fraction is noteworthy compared to the other fractions, presenting a 5% yield compared to the initial algae weight and anticoagulant activity revealed by the activated partial thromboplastin time (APTT) assay (intrinsic/common coagulation pathway). Surprisingly, F44 purification (SP peak P1F44) resulted in prothrombin time (PT) activity (extrinsic coagulation pathway) at a 160 µg/mL, in addition to enhanced APTT activity. The P1F44 anticoagulant activity mechanism was shown to be dependent on two coagulations factors, IIa and Xa, more potent via IIa. Future assessments will be performed to assess this fraction in the medical clinic.

Crosstalk between biological and chemical diversity with cytotoxic and cytostatic effects of Aphanothece halophytica in vitro.

Different solvent extracts from Aphanothece halophytica (A. halophytica) were evaluated for their cytotoxic effects against four human cancer cell lines. The samples demonstrated different percentages of cyanobacteria species populations. The samples containing 100% A. halophytica and 90% A. halophytica showed a significant cytotoxic effect in human breast cancer cells MDA231. The cytostatic effect was demonstrated in MDA231 and human glioblastoma T98G cells regardless of the treatment, resulting in a significant cell cycle arrest in the S phase. The chemical profiles of the extracts were proven to be diverse in qualitative and quantitative compositions. This variability was dependent on the A. halophytica´s abundance in each extract. The 100% A. halophytica extract induced cytotoxic and cytostatic effects in breast cancer cells, and those could be associated with the predominance of fatty acids, hydrocarbons and phthalates, indicating that A. halophytica is an interesting source of novel compound with anticancer effect.

Microwave irradiation is a suitable method for caulerpin extraction from the green algae Caulerpa racemosa (Chlorophyta, Caulerpaceae).

Caulerpin is a bisindolic alkaloid that has been obtained from many species of the genus Caulerpa. The main objective of this paper is to evaluate four extraction methods of caulerpin in the C. racemosa: maceration (DMA), Soxhlet extraction (SOX), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE). The methods were compared through caulerpin content quantified by Ultraviolet-visible (UV-vis) spectrophotometry. The highest extract yield was obtained by SOX but the highest contain of caulerpin was presented in the MAE extract. The caulerpin content was significant different within the extacts by MAE and UAE, it yielded by MAE more than three times as much as UAE. The most efficient caulerpin extraction method had the parameters solvent, temperature and time optimised. Thus, the best conditions were achieved with MAE in ethanol during 7 min at 90 °C. Therefore, this work suggests an improved routine analysis of caulerpin by the green chemistry concept.

Diverse traits of aquatic plants cannot individually explain their consumption by the generalist gastropod Biomphalairia glabrata.

Several experimental studies on aquatic plants have reported the prevalence of chemical defense mechanism against herbivory, as opposed to structural, life-forms or other traits. Here, our laboratory feeding experiments and integrative analysis explored the relationship among palatability (fresh or reconstituted plants used as artificial diet) and various chemical/nutritional traits (i.e., contents of dry mass, ash, nitrogen, protein, and phenols) of diverse aquatic plants and their susceptibility to consumption by the generalist gastropod Biomphalaria glabrata. Biomphalaria glabrata consumed all of the assayed aquatic plants in a hierarchical yet generalized way, with the consumption of fresh plants, their reconstituted forms and defensive properties of lipophilic extracts not being significantly correlated with plant physical or chemical traits to determine the feeding preference of the gastropod. Our results do not reveal a prevalence for a specific plant attribute contributing to herbivory. Instead, they indicate that the susceptibility of aquatic plants to generalist consumers is probably related to a combination of their chemical and physical properties, resulting in moderate grazing rates by generalist consumers.

Differential susceptibility of distinct parts of the aquatic plant Nymphoides humboldtiana to herbivory supports the optimal defense theory.

PREMISE: The optimal defense theory (ODT) predicts that the allocation of chemical defenses in plants will be concentrated in parts or tissues that are of higher fitness value for the individuals that produce them. Chemicals are known to be allocated to certain parts of aquatic plants, and the morphological architecture of Nymphoides humboldtiana, a species that exposes its parts to different environmental factors and consumers, may be an excellent model to evaluate within-plant susceptibility to consumers according to the ODT. METHODS: Under laboratory experimental conditions, we evaluated the defensive properties of extracts from vegetative (leaves, rhizomes, roots) and reproductive (long stem internodes, flowers, fruits) parts of N. humboldtiana against consumption by the generalist herbivorous gastropod Biomphalaria glabrata. Extracts were also subjected to chemical analysis by high-performance liquid chromatography, principal component analysis, and analysis of their relationships to defensive actions. RESULTS: Extracts of all vegetative and internode (reproductive) parts of N. humboldtiana exhibited defensive properties against B. glabrata, but the long stem internodes exhibited the highest percentage of inhibition. Chemical profiles of these parts were qualitatively and quantitatively different, but a major unidentified compound is presumably responsible for the higher defensive property found in internodes. CONCLUSIONS: Our results support the ODT, since chemical defense was more effective in long stem internodes, which have a high fitness value for N. humboldtiana to keep the flowers emerged on the water surface in response to the rapid and dynamic changes in water levels typical of freshwater environments.

The Brazilian octocoral Phyllogorgia dilatata as a source of cytotoxic compounds.

The extensive marine biodiversity has proved to be a promising source of substances with biomedical potential. In this study, the cytotoxicity of the Brazilian octocoral Phyllogorgia dilatata (Gorgoniidae) was evaluated against two tumor cell lines and three bacterial strains. The methanol/dichloromethane crude extract presented no antibacterial activity up to the highest concentration tested (512 µg/mL), however it revealed a noteworthy antiproliferative effect against HCT-116 (80%) and MCF-7 (54%) cell lines at 50 µg/mL. Therefore, guided by the cytotoxic activity, a multistep chemical fractionation of the extract provided the subfraction 5 (PDPH2-5) with IC50 values of 3.18 and 17.80 µg/mL against HCT-116 and MCF-7, respectively. The LC-HRMS/MS analysis of PDPH2-5 showed ions of m/z 219.1742 and 219.1743, characterized as (E,E) and (Z,E) germacrone, after a LC-DAD-SPE/NMR analysis of the hexanic fraction and comparisons of NMR data with the literature. Previously reported assessments to the cytotoxic activity of the (E,E)-diastereoisomer disclosed higher IC50 values than that obtained for the PDPH2-5 fraction, suggesting, herein, a potentiated effect of the diastereoisomeric mixture. Such remark encourage further bioactivity studies with stereoisomer mixtures and reduce the urge for compound isolation.

The sea-hare Aplysia brasiliana promotes induction in chemical defense in the seaweed Laurencia dendroidea and in their congeneric neighbors.

Inducible chemical defenses are more common in temperate seaweeds than tropical ones, and are directly detected by increase of chemical contents, or indirectly by differential consumption of live seaweed tissues or artificial food with algal extracts by herbivores. In general, seaweed-induced chemical defense occur between 11 and 20 days after both simulated/artificial or direct herbivory. Here, we used experimental procedures to assess induced chemical defense in the tropical red seaweed Laurencia dendroidea as response to direct grazing, chemical cues from grazed conspecific neighbors and only presence of herbivores. Chemical defenses were analyzed by detecting the palatability of artificial food containing L. dendroidea extracts offered to Aplysia brasiliana and by comparative analyses of extracts from this seaweed by Gas Chromatography/Mass Spectroscopy, as well as metabolomic data analysis by Principal Component Analysis. Our results revealed that direct grazing by A. brasiliana induced a rapid (after 48 h) response among individuals of L. dendroidea, as did waterborne chemical cues from grazed conspecifics, but the presence of sea hare alone did not elicit a response. Increased resistance to grazing was accompanied by significative changes in sesquiterpene metabolomic chemical profile, revealing that induced defense: may be more widespread among seaweeds, independent of latitude; can involve changes in other classes of substances besides phlorotannins or not only the increase in the content of a single compound; and may be a rapid and ecologically coherent response to consumers. In addition, the importance of incorporating the metabolomic approach when examining inducible chemical defense in seaweeds is also emphasized.

Geographic Distribution of Natural Products Produced by the Red Alga Laurencia dendroidea J. Agardh.

In order to evaluate the chemical diversity of Laurencia dendroidea J. Agardh, a widely distributed seaweed in Brazilian coast, a phytochemical study was carried out with algae collected from six different locations along the Southeast Brazilian coast. Purified compounds were identified by MS and NMR techniques. The chemical profiles of lipophilic extracts were obtained by GC/MS for each population. In total, 15 compounds were described. The sesquiterpene composition accounted for 49 - 63% of the GC/MS chromatogram area. The discrimination of three chemotypes was done by the use of HCA on GC/MS chromatograms. They were also analyzed by the PCA and, together with peak area analysis, it was possible to discriminate all populations by the main variation of elatol, obtusol, rogiolol, and triquinane. The results revealed the high diversity of sesquiterpene composition among populations of L. dendroidea. Curiously, the within and among population variation of elatol and obtusol suggested a biochemical interplay on the content of these compounds. More studies are necessary to understand the patterns of chemical diversity and compound variation within and among populations of L. dendroidea.

Intraspecific variation of meroditerpenoids in the brown alga Stypopodium zonale guiding the isolation of new compounds

Abstract Intraspecific variation on meroditerpenoids production by the brown marine alga Stypopodium zonale at four different populations along the Brazilian coast was analyzed using Principal Component Analysis over high-performance liquid chromatography profiles from algae extracts. The ordination of the samples by the similarities of their chromatographic traits showed the existence of three chemotypes: (i) the populations Búzios and Abrolhos which were characterized by the presence of atomaric acid (1), (ii) the population Atol das Rocas which contained the compound stypoldione (2), and (iii) the population Marataízes which was characterized by other peaks that guided the isolation of three new meroditerpenoids stypofuranlactone (3), 10,18-dihydroxy-5′a-desmethyl-5′-acetylatomaric acid (4), and the 10-keto-10-deisopropyliden-5′a-desmethyl-5′-acetylatomaric acid (5) together with the known compound the 10-keto-10-deisopropyliden-atomaric acid (6). The structures and relative stereochemistry of 3, 4 and 5 were elucidated by NMR and MS techniques. The observed chemical differences among populations of S. zonale can be related to its geographic distribution and can open an avenue to the discovery of new compounds in algae.

Carriebowlinol, an antimicrobial tetrahydroquinolinol from an assemblage of marine cyanobacteria containing a novel taxon.

A combined biodiversity- and bioassay-guided natural products discovery approach was used to explore new groups of marine cyanobacteria for novel secondary metabolites with ecologically relevant bioactivities. Phylogenetic analysis of cyanobacterial collections from Belize revealed a new taxon not previously well explored for natural products. The new alkaloid 5-hydroxy-4-(chloromethyl)-5,6,7,8-tetrahydroquinoline (1), named carriebowlinol, and the known compound lyngbic acid (2) were isolated from a nonpolar extract and identified by NMR and MS techniques. Compounds 1 and 2 inhibited the growth of pathogenic and saprophytic marine fungi, and 1 inhibited the growth of marine bacteria, suggesting an antimicrobial ecological function.

Chemical diversity and antileishmanial activity of crude extracts of Laurencia complex (Ceramiales, Rhodophyta) from Brazil

Chemical profiles of extracts of four species from Laurencia complex (Ceramiales, Rhodophyta) from different populations collected along Southeast Brazilian coast were assessed by High Performance Liquid Chromatography coupled with a Diode Array Detector in order to observe geographic chemical variability. Aiming to evaluate the impact of chemical diversity on potential pharmaceutical uses, the extracts were tested against the promastigote form of Leishmania amazonensis. The most active extracts were submitted to anti-amastigote and cytotoxicity assays. Principal Component Analysis of the chromatograms resulted in four major groups of chemical profiles according to the presence of leishmanicidal chamigranes (-)-elatol and obtusol. The existence of chemotypes, displaying variable pharmacological action, is proposed for the differences observed in L. dendroidea samples. Although all extracts were found active against promastigote form of L. amazonensis, their efficacy was remarkably different and not related to the variation of (-)-elatol and obtusol, which indicates the presence of additional compounds with antileishmanial activity. Moreover, the active extracts also displayed anti-amastigote activity and none of them were considered cytotoxic. The results highlight that the knowledge of chemical geographic variability can be valuable in the search of new antileishmanial compounds from marine sources.

Atividade biológica de metabólitos secundários de algas marinhas do gênero Laurencia / Biological activity of the secondary metabolite from marine algae of the genus Laurencia

As algas vermelhas do gênero Laurencia são conhecidas como uma riquíssima fonte de metabólitos secundários. As principais classes químicas já isoladas são terpenos - sesquiterpenos, diterpenos, triterpenos - e acetogeninas. Estudos apontam que estas substâncias apresentam importantes atividades biológicas, principalmente citotóxica e antibacteriana. Existem ainda alguns poucos trabalhos com informações sobre atividade antiparasitária, antiviral e antifúngica dos metabólitos de Laurencia. O forte perfil bioativo observado em alguns estudos sugere que alguns destes metabólitos podem ser futuramente utilizados como fármacos ou como protótipos de novos agentes antitumorais e antibacterianos. Muitos dos metabólitos de Laurencia ainda não foram avaliados sob o ponto de vista farmacológico, o que representa um grande potencial a ser explorado por diversos campos da farmacologia.

The red alga of the genus Laurencia is known as an endless source of metabolites. The main substances produced are sesquiterpenes, diterpenes, triterpenes and acetogenins. Many studies reveal that these metabolites are active, especially cytotoxic and antibacterial. There are few other studies that describe the antiparasital, antifungal and antiviral actions of the metabolites produced by the alga of the genus Laurencia. The results observed in some studies suggest that these metabolites may be used in the future as drugs or lead compounds as antitumoral and/or antibacterial agents. Many metabolites have not been submitted to pharmacological studies which still represents an area to be explored.

Induction of attachment of the Mussel Perna perna by natural products from the brown seaweed Stypopodium zonale.

Marine invertebrates settle, attach, and/or metamorphose in response to signals from several sources, including seaweeds. In response to the aquaculture challenge of producing constant numbers of juveniles from cultured species, natural inducers have been screened for their ability to improve those processes. However, few chemical inducers of attachment of invertebrates have been identified, and even less of these were secondary metabolites. The goal of this work was to isolate the natural products responsible for induction activity using bioassay-guided fractionation of the organic extract of the brown seaweed Stypopodium zonale and the attachment of juveniles of the common brown mussel, Perna perna, as a model. The meroditerpene epitaondiol, identified by comparison of spectral data with the literature, promoted as much as 4.7 times more mussel attachment compared to controls at the natural concentration found in this alga (0.041% of the crude extract or 0.012% of algal dry weight). This is the first report showing that a seaweed produces terpenoid compounds as cues for invertebrate attachment, and future studies evaluating this action on settlement of mussels in the field are expected to improve aquaculture technology by increasing mussel spat production.

Is the mussel test a good indicator of antifouling activity? A comparison between laboratory and field assays.

Current antifouling technologies rely on metal-based paints, but due to their toxicity, an expected worldwide ban of organotin-containing paints is now prompting the quest for safe and effective alternatives. One of these is antifouling coatings whose active components are naturally occurring compounds in marine organisms. A number of laboratory bioassays has been designed to search for antifouling compounds. However, there is no evidence to date that these assays provide results reproducible through ecologically realistic field experiments. Natural concentrations of the extracts from the Brazilian seaweeds Laurencia obtusa and Stypopodium zonale were tested in the laboratory through the 'mussel test' and in the field through the 'phytagel method' in order to compare the efficiency of these methods in assessing antifouling activity. L. obtusa extract significantly inhibited fouling in both the laboratory and field assays, while S. zonale stimulated fouling in both assays. Major compounds from the extracts were identified. The findings suggest that the 'mussel test' is a reliable time and cost-saving screening method for antifouling substances, although field assays are more sensitive for detection of their activity spectrum.