Stéphane Cérantola

Phlorotannins in sargassaceae species from brittany (France): Interesting molecules for ecophysiological and valorisation purposes

Phlorotannins are metabolites synthesised by brown algae to protect against environmental stresses. Only a few studies presented structural elucidation of native molecules extracted from macroalgae, contrary to previous published studies, which characterised acetylated phlorotannins. The present work introduces quantitative and qualitative studies to characterise phlorotannins from the eight species of Sargassaceae settled in Brittany, using a colorimetric method used for quantification of total phenolic content (TPC), and one- and two-dimensional nuclear magnetic resonance (NMR) (1H, heteronuclear multiple bond correlation) together with in vivo NMR (high-resolution magic-angle spinning (HR-MAS) NMR) analyses, with the aim to elucidate structural and fingerprint of phlorotannins signals. Halidrys siliquosa, which settles in low tide rock pools, exhibited high TPC (17.77±1.68% dry weight (DW)) while the other species showed lower TPC (ranged from 0.85±0.11% DW for Cystoseira baccata growing in low tide rock pools to 5.53±0.63% DW in Cystoseira humilis growing in high tide rock pools). No relation between TPC and the position of the algae along the shore could be highlighted. Fingerprints using NMR HR-MAS were useful to compare the eight species in terms of phenolic content and in terms of phenolic signals as the species from Brittany produced different phlorotannins. Our study demonstrated that Cystoseira tamariscifolia produced the monomer (phloroglucinol), C. humilis, phloroglucinol and a phlorethol, C. baccata and Cystoseira nodicaulis produced many compounds, i.e. traces of phloroglucinol together with fucols, phlorethols, and fucophlorethols and finally fuhalols were identified in Bifurcaria bifurcata and H. siliquosa. The putative ecophysiological roles of these phlorotannins from Sargassaceae are discussed, together with their potential bioactivities.

Sunscreen, antioxidant, and bactericide capacities of phlorotannins from the brown macroalga Halidrys siliquosa

The present study focused on a brown macroalga (Halidrys siliquosa), with a particular emphasis on polyphenols and their associated biological activities. Two fractions were obtained by liquid/liquid purification from a crude hydroethanolic extract: (i) an ethyl acetate fraction and (ii) an aqueous fraction. Total phenolic contents and antioxidant activities of extract and both fractions were assessed by in vitro tests (Folin–Ciocalteu test, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power assay, superoxide anion scavenging assay, and β-carotene–linoleic acid system). For the most active fraction, i.e., the ethyl acetate fraction, the oxygen radical absorbance capacity (ORAC) value, antibacterial activities, and sunscreen potential (Sun Protection Factor and UV-A-Protection Factor) were tested in vitro. A high correlation found between antioxidant activities and total phenolic content was interpreted as the involvement of polyphenolic compounds in antioxidant mechanisms. Interestingly, the ethyl acetate fraction appeared to be a broad-spectrum UV absorber and showed a strong bactericidal activity against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. In this fraction, four phenolic compounds (trifuhalols and tetrafuhalols and, for the first time, diphlorethols and triphlorethols) were identified using 1D and 2D nuclear magnetic resonance (NMR) and MS analysis. These findings are promising for the use of H. siliquosa, abundant in Brittany, as a valuable source of photoprotectant molecules for sunscreen and cosmetic applications.

HRMAS NMR as a tool to study metabolic responses in heart clam Ruditapes decussatus exposed to Roundup

The essence of this study was to investigate the metabolic responses of heart tissues of carpet-shell clam Ruditapes decussatus after exposure to two doses (0.2 and 1 g/L) of Roundup(®) during 24 and 72 h. The main metabolic changes after Roundup(®) exposure were related to disturbance in energy metabolism and metabolic biomarkers such as alanine, succinate, acetate and propionate, suggesting the occcurence of anaerobiosis and the impairment of oxydative metabolism. Results showed also that peak intensities of amino acids used as biomarker of anaerobiosis in molluscs are time and dose dependent. In the opposite, phosphoarginine and ATP level are dependent to Roundup(®) concentration rather than to the time of exposure. We suggest that changes in energy demands require adjustements in the forward arginine kinase reaction rate. Therefore, the results demonstrate the high applicability of HRMAS NMR to elucidate the mechanism of toxicity of Roundup(®). In addition, (31)P HRMAS NMR appeared to be an effective and simple method to follow bioaccumulation of Roundup(®) formulation.

NMR use to quantify phlorotannins: The case of Cystoseira tamariscifolia, a phloroglucinol-producing brown macroalga in Brittany (France)

Among the most renowned natural products from brown algae, phlorotannins are phloroglucinol polymers that have been extensively studied, both for their biotechnological potential and their interest in chemical ecology. The accurate quantification of these compounds is a key point to understand their role as mediators of chemical defense. In recent years, the Folin-Ciocalteu assay has remained a classic protocol for phlorotannin quantification, even though it frequently leads to over-estimations. Furthermore, the quantification of the whole pool of phlorotannins may not be relevant in ecological surveys. In this study, we propose a rapid (1)H qNMR method for the quantification of phlorotannins. We identified phloroglucinol as the main phenolic compound produced by the brown macroalga Cystoseira tamariscifolia. This monomer was detected in vivo using (1)H HR-MAS spectroscopy. We quantified this molecule through (1)H qNMR experiments using TSP as internal standard. The results are discussed by comparison with a standard Folin-Ciocalteu assay performed on purified extracts. The accuracy and simplicity of qNMR makes this method a good candidate as a standard phlorotannin assay.

Characterization and anti-biofilm activity of extracellular polymeric substances produced by the marine biofilm-forming bacterium Pseudoalteromonas ulvae strain TC14

Abstract This study investigated soluble (Sol-EPS), loosely bound (LB-EPS), and tightly bound extracellular polymeric substances (TB-EPS) harvested from biofilm and planktonic cultures of the marine bacterium Pseudoalteromonas ulvae TC14. The aim of the characterization (colorimetric methods, FTIR, GC-MS, NMR, HPGPC, and AFM analyses) was to identify new anti-biofilm compounds; activity was assessed using the BioFilm Ring Test®. A step-wise separation of EPS was designed, based on differences in water-solubility and acidity. An acidic fraction was isolated from TB-EPS, which strongly inhibited biofilm formation by marine bacterial strains in a concentration-dependent manner. The main constituents of this fraction were characterized as two glucan-like polysaccharides. An active poly(glutamyl-glutamate) fraction was also recovered from TB-EPS. The distribution of these key EPS components in Sol-EPS, LB-EPS, and TB-EPS was distinct and differed quantitatively in biofilm vs planktonic cultures. The anti-biofilm potential of the fractions emphasizes the putative antifouling role of EPS in the environment.

Spatiotemporal variations of diterpene production in the brown macroalga Bifurcaria bifurcata from the western coasts of Brittany (France)

Bifurcaria bifurcata, a temperate brown macroalga, is known to show spatial fluctuations in its diterpene content along the northwestern coasts of France (Brittany). In the aim to identify environmental factors which could influence the occurrence of a particular chemical type, several populations of B. bifurcata were collected in summer 2009 and winter 2010. Their chemical composition was studied through thin layer chromatography (TLC) and nuclear magnetic resonance (NMR) analyses. Results showed that specific diterpenes are biosynthesized depending on seasons and abiotic factors, such as hydrodynamism or substratum. Exposed sites were characterized by thalli of B. bifurcata producing two main diterpenoids, bifurcane, and eleganediol, whereas thalli from sheltered sites showed crude extracts containing a major diterpene, eleganolone. On these last sites, another diterpene (bifurcanone) is only expressed in winter and was thus considered as a seasonal chemomarker. The term “chemotype” applied to a population is proposed and discussed.

Monohalogenated maleimides as potential agents for the inhibition of Pseudomonas aeruginosa biofilm

New monohalogenated maleimide derivatives (with bromine, chlorine or iodine) were synthesized to test the effect of halogen atoms in inhibiting the formation of Pseudomonas aeruginosa biofilm. The evaluation of their biological activities clearly defines a structure–activity relationship. In this study, the bactericidal action of the three compounds was observed at the concentration range 0.3–5.0 mM on Luria-Bertani agar plates. The halogen atom of these molecules was critical in modulating the antibacterial activity, with a slightly higher effectiveness for chlorine. Confocal laser scanning microscopy was used to examine P. aeruginosa biofilms cultivated in flow cells. At concentration as low as 40 μM, the bromine and iodine compounds displayed a total inhibition towards the formation of bacterial biofilm. At this concentration, the bacterial attachment to glass surfaces was strongly affected by the presence of bromine and iodine whereas the chlorine derivative behaved as a bactericidal compound. A bioluminescent reporter strain was then used to detect the effect of the chemically synthesized maleimides on quorum sensing (QS) in P. aeruginosa. At the concentration range 10–100 μM, bioluminescence assays reveal that halogenated maleimides were able to interfere with the QS of the bacterium. Although the relationship between the weak inhibition of cell-to-cell communication (15–55% of the signal) and the high inhibition of biofilm formation has not been elucidated clearly, the results demonstrate that bromo- and iodo-N-substituted maleimides bromine and iodine may be used as new potent inhibitors that control bacterial biofilms.

Isolation of turbinaric acid as a chemomarker of Turbinaria conoides (J. Agardh) Kützing from South Pacific Islands

Several species of the genus Turbinaria coexist along the coasts of islands in the Indian and Pacific Oceans. Among these brown algae, Turbinaria ornata and T. conoides are sister species that are difficult to differentiate using exclusively morphological characters. Based on in vivo nuclear magnetic resonance and chromatographic techniques, i.e., liquid and gas chromatography‐mass spectrometry analysis, combined with phylogenetic data, we successfully identified turbinaric acid in T. conoides samples from several Indian and Pacific Ocean islands. This nonvariable discriminant molecule was only identified in T. conoides specimens, but not in the two allied species T. ornata and T. decurrens. Results are discussed with regard to turbinaric acid as an interesting chemomarker isolated from T. conoides and the rapid discrimination of Turbinaria specimens using chemical assays.

HRMAS NMR Analysis of Algae and Identification of Molecules of Interest via Conventional 1D and 2D NMR: Sample Preparation and Optimization of Experimental Conditions

Nuclear magnetic resonance (NMR) has become an astounding tool for molecular characterization. Thanks to the development of probes and the increase of magnetic field, NMR has entered the field of biology and facilitated the identification of natural compounds. Indeed, this nondestructive NMR tool makes possible the complete characterization of less and less quantities of material via 1D and 2D sequences on many nuclei (e.g., (1)H, (13)C, (31)P, (15)N). More recently, the development of high-resolution magic-angle spinning (HRMAS) probes have permitted direct analysis of living tissue (e.g., a piece of algae) without prior extraction providing information on both the total content and the ratio of different molecules within the sample; thus HRMAS facilitates a wide range of analyses, such as species differentiation or studies of metabolomics according to various environmental or experimental conditions. This chapter describes the specific sample preparation, based on an algal sample or extract, required for all NMR analyses in order to optimize the NMR response and obtain the most valuable information.