A Characterization of Biological Activities and Bioactive Phenolics from the Non-Volatile Fraction of the Edible and Medicinal Halophyte Sea Fennel (Crithmum maritimum L.).

Although the biochemical composition and biological properties of the volatile fraction of the halophyte sea fennel (Crithmum maritimum L.) have been largely described, little is known about its polar constituents and bioactivities. Here, a hydromethanolic extract of Crithmum maritimum (L.) leaves was fractionated, and the fractions were evaluated in vitro for antioxidant (using DPPH, ABTS, and FRAP bioassays), anti-inflammatory (inhibition of NO production in RAW 264.7 macrophages), antidiabetic (alpha-glucosidase inhibition), neuroprotective (inhibition of acetylcholinesterase), and skin-protective (tyrosinase and melanogenesis inhibitions) activities. Polar fractions of the extract were rich in phenolics and, correlatively, displayed a strong antioxidant power. Moreover, fractions eluted with MeOH20 and MeOH80 exhibited a marked inhibition of alpha-glucosidase (IC50 = 0.02 and 0.04 mg/mL, respectively), MeOH60 fractions showed a strong capacity to reduce NO production in macrophages (IC50 = 6.4 µg/mL), and MeOH80 and MeOH100 fractions had strong anti-tyrosinase activities (630 mgKAE/gDW). NMR analyses revealed the predominance of chlorogenic acid in MeOH20 fractions, 3,5-dicaffeoylquinic acid in MeOH40 fractions, and 3-O-rutinoside, 3-O-glucoside, 3-O-galactoside, and 3-O-robinobioside derivatives of quercetin in MeOH60 fractions. These compounds likely account for the strong antidiabetic, antioxidant, and anti-inflammatory properties of sea-fennel polar extract, respectively. Overall, our results make sea fennel a valuable source of medicinal or nutraceutical agents to prevent diabetes, inflammation processes, and oxidative damage.

An Eco-Friendly Extraction and Purification Approach for Obtaining Active Ingredients for Cosmetics from Two Marine Brown Seaweeds.

Brown seaweeds are attracting attention due to their richness in bioactive compounds, in particular, their phlorotannins. We present here a case study of two Fucales, Ascophyllum nodosum and Halidrys siliquosa, sustainably collected, to produce active polyphenols for the cosmetics sector. Phenolic contents of crude extracts, obtained by Accelerated Solvent Extraction (ASE), were more elevated in H. siliquosa at 100.05 mg/g dry weight (DW) than in A. nodosum (29.51 mg/g DW), considering 3 cycles with cell inversion. The temperature of extraction for a high phenolic content and high associated antioxidant activities close to positive controls was 150 °C for both algae and the use of only one cycle was enough. A semi-purification process using Solid-phase Extraction (SPE) was carried out on both ASE crude extracts (one per species). The majority of phlorotannins were found in the ethanolic SPE fraction for A. nodosum and the hydroethanolic one for H. siliquosa. The SPE process allowed us to obtain more concentrated fractions of active phenolic compounds (×1.8 and 2 in A. nodosum and H. siliquosa, respectively). Results are discussed in regard to the exploitation of seaweeds in Brittany and to the research of sustainable processes to produce active natural ingredients for cosmetics.

Sulfated Polysaccharides from Seaweed Strandings as Renewable Source for Potential Antivirals against Herpes simplex Virus 1.

Herpes simplex virus 1 (HSV-1) remains a prominent health concern widespread all over the world. The increasing genital infections by HSV-1 that might facilitate acquisition and transmission of HIV-1, the cumulative evidence that HSV-1 promotes neurodegenerative disorders, and the emergence of drug resistance signify the need for new antiviral agents. In this study, the in vitro anti-herpetic activity of sulfated polysaccharides (SPs) extracted by enzyme or hot water from seaweeds collected in France and Mexico from stranding events, were evaluated. The anti-herpetic activity evaluation of the semi-refined-polysaccharides (sr-SPs) and different ion exchange purified fractions showed a wide range of antiviral activity. Among them, the sr-SPs from the Rhodophyta Halymenia floresii showed stronger activity EC50 0.68 µg/mL with SI 1470, without cytotoxicity. Further, the antiviral activity of the sr-SPs evaluated at different treatment schemes showed a high EC50 of 0.38 µg/mL during the viral adsorption assays when the polysaccharide and the virus were added simultaneously, whilst the protection on Vero cell during the post-infection assay was effective up to 1 h. The chemical composition, FTIR and 1H NMR spectroscopic, and molecular weights of the sr-SPs from H. floresii were determined and discussed based on the anti-herpetic activity. The potential utilization of seaweed stranding as a source of antiviral compounds is addressed.

First report on phytochemical investigation, antioxidant and antidiabetic activities of Helianthemum getulum.

A new flavonoid, 5,7,2',4',5'-pentahydroxyflavone 3-O-ß-D-galactopyranoside (12) and twelve known derivatives: an aryltetralin-lignan (3), seven flavonoids (4-5, 7-10, 13) and four phenolic acids (1-2, 6, 11) have been isolated from the aerial parts of Helianthemum getulum Pomel. (Cistaceae family) an endemic species to the septentrional Sahara that is being studied for the first time. Structure elucidation of the isolated compounds was established by means of spectroscopic methods especially NMR and Mass Spectrometry. In vitro antioxidant (DPPH, ABTS, GOR and CUPRAC assays) and antidiabetic (micro-dilution method) activities of the crude extract, fractions and isolated compounds were performed. The new flavonol (12) and Compounds (2, 3, 7, 9) were found to be the most active, some of them exhibiting better activity than the antioxidant standards. Compounds 7, 9 and 3 showed higher α-glucosidase inhibitory activity compared to standard acarbose (IC50= 2.70 ± 0.03 µM, 3.09 ± 0.03 µM, 37.28 ± 1.20 µM and 275.43 ± 1.59 µM, respectively).

Phlorotannin and Pigment Content of Native Canopy-Forming Sargassaceae Species Living in Intertidal Rockpools in Brittany (France): Any Relationship with Their Vertical Distribution and Phenology?

Five native Sargassaceae species from Brittany (France) living in rockpools were surveyed over time to investigate photoprotective strategies according to their tidal position. We gave evidences for the existence of a species distribution between pools along the shore, with the most dense and smallest individuals in the highest pools. Pigment contents were higher in lower pools, suggesting a photo-adaptive process by which the decreasing light irradiance toward the low shore was compensated by a high production of pigments to ensure efficient photosynthesis. Conversely, no xanthophyll cycle-related photoprotective mechanism was highlighted because high levels of zeaxanthin rarely occurred in the upper shore. Phlorotannins were not involved in photoprotection either; only some lower-shore species exhibited a seasonal trend in phlorotannin levels. The structural complexity of phlorotannins appears more to be a taxonomic than an ecological feature: Ericaria produced simple phloroglucinol while Cystoseira and Gongolaria species exhibited polymers. Consequently, tide pools could be considered as light-protected areas on the intertidal zone, in comparison with the exposed emerged substrata where photoprotective mechanisms are essential.

Evaluation of the Antioxidant, Anti-Inflammatory and Cytoprotective Activities of Halophyte Extracts against Mycotoxin Intoxication.

Twelve halophyte species belonging to different families, widely represented along French Atlantic shoreline and commonly used in traditional medicine, were screened for protective activities against mycotoxins, in order to set out new promising sources of natural ingredients for feed applications. Selected halophytic species from diverse natural habitats were examined for their in vitro anti-mycotoxin activities, through viability evaluation of Madin-Darby Bovine Kidney (MDBK) and intestinal porcine enterocyte (IPEC-J2) cell lines. Besides, the in vitro antioxidant activities of plant extracts were assessed (total antioxidant and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging bioassays). Of the 12 species, Galium arenarium, Convolvulus soldanella and Eryngium campestre exhibited the most protective action on MDBK and IPEC-J2 cells against zearalenone (ZEN) or T2 toxin contamination (restoring about 75% of cell viability at 10 µg·mL-1) without inflammation response. They also had strong antioxidant capacities (Inhibitory concentration of 50% (IC50) < 100 µg·mL-1 for DPPH radical and total antioxidant capacity (TAC) of 100 to 200 mg Ascorbic Acid Equivalent (AAE)·g-1 Dry Weight), suggesting that cell protection against intoxication involves antioxidant action. A bio-guided study showed that fractions of G. arenarium extract protect MDBK cells against T2 or ZEN toxicity and several major compounds like chlorogenic acid and asperuloside could be involved in this protective effect. Overall, our results show that the halophytes G. arenarium, C. soldanella and E. campestre should be considered further as new sources of ingredients for livestock feed with protective action against mycotoxin intoxication.

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

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.

A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid.

GY785 is an exopolysaccharide produced by a mesophilic bacterial strain Alteromonas infernus discovered in the deep-sea hydrothermal vents. GY785 highly sulfated derivative (GY785 DRS) was previously demonstrated to be a promising molecule driving the efficient mesenchymal stem cell chondrogenesis for cartilage repair. This glycosaminoglycan- (GAG-) like compound was modified in a classical solvent (N,N'-dimethylformamide). However, the use of classical solvents limits the polysaccharide solubility and causes the backbone degradation. In the present study, a one-step efficient sulfation process devoid of side effects (e.g., polysaccharide depolymerization and/or degradation) was developed to produce GAG-like derivatives. The sulfation of GY785 derivative (GY785 DR) was carried out using ionic liquid as a reaction medium. The successful sulfation of this anionic and highly branched heteropolysaccharide performed in ionic liquid would facilitate the production of new molecules of high specificity for biological targets such as tissue engineering or regenerative medicine.

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.

The marine bacteria Cobetia marina DSMZ 4741 synthesizes an unexpected K-antigen-like exopolysaccharide.

We have studied the exopolysaccharide produced by Cobetia marina DSMZ 4741, a marine bacterium isolated from coastal seawater. This strain is able to produce a polysaccharide in presence of carbon sources as glucose, mannitol and alginate. The maximum production occurs in aerobic condition, during the end of the exponential phase. The polymer is a non-viscous, acidic heteropolysaccharide of 270kDa constituted of a repeating unit of: This kind of chemical structure is generally related to K-antigen polysaccharide of pathogenic Escherichia coli strains. This is the first time this type of EPS is described from a marine bacterium. Moreover the polysaccharide exhibits a pyruvate substitution on its 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue never encountered before. The discovery of such an unexpected EPS with high biotechnological potential is a new incentive for a better exploration of bioactive marine resources.

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.

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.

Antiviral activities of sulfated polysaccharides isolated from Sphaerococcus coronopifolius (Rhodophytha, Gigartinales) and Boergeseniella thuyoides (Rhodophyta, Ceramiales).

Water-soluble sulfated polysaccharides isolated from two red algae Sphaerococcus coronopifolius (Gigartinales, Sphaerococcaceae) and Boergeseniella thuyoides (Ceramiales, Rhodomelaceae) collected on the coast of Morocco inhibited in vitro replication of the Human Immunodeficiency Virus (HIV) at 12.5 µg/mL. In addition, polysaccharides were capable of inhibiting the in vitro replication of Herpes simplex virus type 1 (HSV-1) on Vero cells values of EC50 of 4.1 and 17.2 µg/mL, respectively. The adsorption step of HSV-1 to the host cell seems to be the specific target for polysaccharide action. While for HIV-1, these results suggest a direct inhibitory effect on HIV-1 replication by controlling the appearance of the new generations of virus and potential virucidal effect. The polysaccharides from S. coronopifolius (PSC) and B. thuyoides (PBT) were composed of galactose, 3,6-anhydrogalactose, uronics acids, sulfate in ratios of 33.1, 11.0, 7.7 and 24.0% (w/w) and 25.4, 16.0, 3.2, 7.6% (w/w), respectively.

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 microM, 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 microM, 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.

New antibacterial and cytotoxic activities of falcarindiol isolated in Crithmum maritimum L. leaf extract.

Bioassay-guided fractionation of a chloroformic extract obtained from Crithmum maritimum leaves led to the chemical isolation of falcarindiol, a polyacetylene widely distributed within the Apiaceae family. Structure of this compound was confirmed by NMR and activity was tracked using a screening microplate bioassay. Falcarindiol strongly inhibited the growth of Micrococcus luteus and Bacillus cereus, with a minimum inhibitory concentration (MIC) value of 50 microg ml(-1). Moreover, this compound showed cytotoxicity against IEC-6 cells with an IC50 value of 20 microM after 48 h of exposition. These results suggest that Crithmum maritimum could be potentially used in food manufactures and cosmetology as preservative agents and biopesticides, or in medicine as new antibiotics, confirming the interest in studying halophytic species as sources of bioactive compounds.

Impact of the salt stress on the photosynthetic carbon flux and 13C-label distribution within floridoside and digeneaside in Solieria chordalis.

The flux of photosynthetic carbon used in the synthesis of low-molecular weight carbohydrates (digeneaside and floridoside) was investigated by (13)C and (1)H NMR spectroscopy in samples of the red seaweed, Solieria chordalis, incubated at different salinities (22, 34 and 50psu). Carbohydrates were labelled, by pulse-chase, with the stable isotope (13)C from NaH(13)CO(3). In vivo NMR analyses carried out with a cryogenic probe optimised for (13)C detection were performed directly on the living algal tissues to evidence the labelling of the carbohydrates with neither preliminary extraction nor purification step(s). The isotopic enrichment of each compound was determined by high-resolution (1)H and (13)C NMR spectroscopy. These analyses evidenced different orientations of the flux of the photosynthetic carbon in the algae according to the salt stress. At normal and low salinities, the photosynthetic carbon flux was responsible of 70% and 67% of the floridoside synthetized during the pulse period, respectively, whereas it was only of 30% in the thalli exposed to the high salinity, meaning a biosynthesis of high floridoside amount from endogen source leading to the osmotic regulation. Under normal and hyper-osmotic conditions, the stock of floridoside was used for cellular needs during the chase period, whereas it was not under hypo-osmotic conditions. The characterization of isotopomers composition of floridoside and digeneaside and the analysis of adjacent (13)C-labelling gives much more details on the effects of salinity on the metabolic pathways leading to the synthesis or the degradation of these molecules. High turnover of floridoside was evidenced at normal salinity during the chase period and products issued from the degradation of floridoside would not be used for the novo biosynthesis. That suggests that synthesis and degradation of floridoside may be realized in two different cellular compartments. The presence of more numerous (13)C-(13)C blocks in the carbon skeleton of the molecules from the up salt stressed thalli than in those from no salt stressed algae, concomitant with a slower degree of isotopic enrichment of the molecule, provided evidence that the two metabolic pathways (endogen and photosynthetic) may not share the precursor molecules involved in the floridoside synthesis and that these two routes may be totally separate until the constitution of floridoside molecule.

General occurrence of the glucosinolate glucocochlearin within the Cochlearia genus.

A natural compound, glucocochlearin, was isolated from the aerial parts of 10 different Cochlearia species. The purification of this compound was achieved through HPLC. The identity of the product was established mainly on the basis of spectroscopic NMR (1H, 13C, COSY, TOCSY, HMQC, HMBC, J-MOD) and high resolution mass spectroscopy data. This compound can be considered as a chemomarker of the genus Cochlearia.

NMR characterisation of inulin-type fructooligosaccharides as the major water-soluble carbohydrates from Matricaria maritima (L.).

By use of 1H and 13C NMR spectroscopy including 2D 1H,1H DQF-COSY/TOCSY and 1H,13C HMQC/HMBC experiments, the main water-soluble carbohydrate components extracted from leaves of Matricaria maritima were identified as oligofructans composed of a linear chain of (2-->1)-linked beta-D-fructofuranosyl residues specifying an inulin-type structure. Alpha-D-Glcp-(1-->2)-[beta-D-Fruf-(2-->1)-beta-D-Frucf]n-(2-->1)-beta-D-Fruf.

Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin. Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains.

The attenuated strain of Mycobacterium bovis Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy, is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. Mycobacterium tuberculosis and other mycobacterial species produce a glycogen-like alpha-glucan composed of repeating units of 4-linked glucosyl residues substituted at some 6 positions by short oligoglucosyl units that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of alpha-glucans purified from the extracellular materials and boiling water extracts of three vaccine substrains. The polysaccharides were purified, and their structural features were established by mono- and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like alpha-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted alpha-glucans in mycobacteria.