Large-scale bioprospecting of cyanobacteria, micro- and macroalgae from the Aegean Sea

Sofia Montalvão1, Zeliha Demirel2, Prabha Devi3, Valter Lombardi4, Vesa Hongisto5, Merja Perälä5, Johannes Hattara6, Esra Imamoglu2, Supriya Shet Tilvi3, Gamze Turan7, Meltem Conk Dalay2, Päivi Tammela1, *

1Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy,

University of Helsinki, P.O. Box 56, FI-00014 Helsinki, Finland; 2Bioengineering Department,

Engineering Faculty, Ege University, 35100, Bornova, Izmir, Turkey; 3CSIR-National Institute of

Oceanography, Dona Paula, 403004, Goa, India; 4Euroespes Biotechnology, Department of

Cellular Immunology, 15165 Bergondo, A Coruña, Spain; 5VTT Technical Research Centre of

Finland, Knowledge Intensive Products and Services, FI-20520 Turku, Finland; 6VTT Technical

Research Centre of Finland, Solutions for natural resources and environment, FI-20520 Turku,

Finland; 7Aquaculture Department, Fisheries Faculty, Ege University, 35100, Bornova, Izmir,

Turkey

*Corresponding author. E-mail address: [email protected], Phone number: +358 2941 59628

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ABSTRACT

Marine organisms constitute approximately one half of the total global biodiversity, being rich reservoirs of structurally diverse biofunctional components. The potential of cyanobacteria, micro- and macroalgae as sources of antimicrobial, antitumoral, anti-inflammatory, and anticoagulant compounds has been reported extensively. Nonetheless, biological activities of marine fauna and flora of the Aegean Sea have remained poorly studied when in comparison to other areas of the Mediterranean Sea. In this study, we screened the antimicrobial, antifouling, anti-inflammatory and anticancer potential of in total 98 specimens collected from the Aegean Sea. Ethanol extract of diatom Amphora cf capitellata showed the most promising antimicrobial results against Candida albicans while the extract of diatom Nitzschia communis showed effective results against Gram-positive bacterium, S. aureus. Extracts from the red alga Laurencia papillosa and from three Cystoseira species exhibited selective antiproliferative activity against cancer cell lines and an extract from the brown alga Dilophus fasciola showed the highest anti- inflammatory activity as measured in primary microglial and astrocyte cell cultures as well as by the reduction of proinflammatory cytokines. In summary, our study demonstrates that the Aegean Sea is a rich source of species that possess interesting potential for developing industrial applications.

Keywords

Antimicrobial, antiproliferative, antifouling, anti-inflammatory, marine algae

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INTRODUCTION Bioprospecting, a tool for biodiversity conservation, can be defined as the collection of biological material and analysis of its properties and/or its molecular, biochemical or genetic content for a purpose of developing a commercial product. However, special care must be given to ethical issues and conservation policies [1–3]. Covering more than 70% of world’s surface and with still many unknown habitats and species, the oceans and seas play an important role in the search for new solutions for improving human health. In recent years, many reports have been published about the biological activities of marine species (e.g., cyanobacteria, macro- and microalgae) and compounds isolated thereof [4–11].

The Aegean Sea is a saltwater body in the northeast part of the Mediterranean Sea. Being adjacent to the Mediterranean – a marine biodiversity hotspot [12] – makes this embayment an important site to study marine resources. The Aegean Sea has endemic and rare species. The season in the Aegean Sea is characterized by the presence of two distinct periods (summer and winter) with spring and autumn relatively short and transitional. However, the Aegean Sea has received little attention in bioprospecting studies, despite its economic and cultural importance.

Marine macroalgae, macroscopic and multicellular seaweeds, can be classified into three groups based primarily on their photosynthetic pigmentation: green (Chlorophyta), red (Rhodophyta) and brown (Phaeophyta) [13]. Each group is rich in a certain type of sulphated polysaccharide: ulvan (mainly in green algae), carrageenan (mainly in red algae) or fucoidan (mainly in brown algae). Many studies have shown that seaweeds provide a rich source of natural bioactive compounds, which are known to display antibacterial [14], antitumoral [15], antiproliferative [16], antioxidant

[16–18], and anti-inflammatory [16,19] activities. For example, Abou Zeid et al. showed that polysaccharides of macroalgae Dictyopteris membranacea exhibited interesting antimicrobial and antitumoral activities [20].

Microalgae (or diatoms) are microscopic and unicellular, and normally classified not only by the presence or absence of cell nucleus, by the presence of flagella and cycle of life but also, as seaweed, by their photosynthetic pigmentation. Extracts from diatoms have been studied, for

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example, for their apoptotic effects [21,22] and biochemical composition [23]. However, many aspects of diatom’s metabolism and production of natural compounds remain unknown.

Macro- and microalgae are well recognized as valuable resources for industrial purposes. Cultivation of microalgae is known to be one of the most profitable business in the biotech industry, being used as nutritional supplements [24,25], for CO2 fixation [26], in cosmetics[27,28], food colorants [29], and as biofuel [30], among others. Macroalgae, crucial primary producer in oceanic aquatic food webs, is equally important ecologically and commercially. Macroalgae applications cover human food [31,32], pharmaceuticals [33,34], cosmeceuticals [35] and energy production [36,37].

The objective of this work was to carry out large-scale screening of antimicrobial, antifouling, anti-inflammatory and anticancer properties of in total 98 specimens collected from the Aegean Sea and to evaluate their potential from biosprospecting perspective.

MATERIALS AND METHODS

Sample collection and preparation

Collection of cyanobacteria, and micro- and macroalgae Altogether 98 cyanobacteria, micro- and macroalgae specimens were collected from the Aegean Sea, Turkey. Details on the collected species, collection sites and identification are provided as Supplementary Information (Table A.1). Macroalgae were collected from the seashore by hand picking while cyanobacteria and microalgae were collected by plankton net (mesh size 20 µm). Samples were packed into clean nylon bags or polyethylene bottles, placed on ice, and taken to the laboratory within 48 hours. Microalgae and cyanobacteria species were checked and identified under microscope, and then diluted and plated onto plates to prepare monoalgal cultures.

Isolation and purification of cyanobacteria and microalgae species Collected sample (1 ml) was inoculated into 9 ml of appropriate sterilized medium in a 15-ml tube. The tube was incubated for 7 days at 25°C at the light intensity of 30 µmol photons m-2 s-1.

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Isolation was accomplished by streaking the sample across the agar surface. Isolated colonies were picked from the agar plate with disposable loop and then both re-streaked on a new agar plate and rinsed in appropriate liquid medium to suspend the cells. Isolates were incubated at 25°C at light intensity of 40 µmol photons m-2 s-1 in 250-ml Erlenmeyer flasks for 14 days. Isolated species were added to the Ege University Microalgae Culture Collection [38].

Batch cultivation of cyanobacteria and microalgae Cyanobacteria and microalgae samples were monoalgal (non-axenic) and cultured in appropriate media at 22±2 °C under continuous illumination (100 µmol photons m-2 s-1) in 2-l sterile bottles for 23 days. Illumination was provided by standard cool white fluorescent lamps (18 W) from one side of the flask. Irradiance was measured in the center of the bottle with a quantum meter (Lambda L1-185). Air was continuously supplied to the culture at a flow rate of 1 l/min (1.25 vvm) by air pump.

Preparation of extracts After the cultivation period of microalgae strains, cells from the culture broth (> 5 l) were collected and separated using GEA Westafalin separator mineral oil systems GmbH (30,000 rpm, 30 min, 25°C) and the supernatant was removed. If the volume of culture broth was less than 5 l, the cells were separated by using Heichman centrifuge (5,000 rpm, 4 min, 18°C) and the supernatant was removed. Collected cells were washed with distilled water and then dried using Alpha 1-2 /LD plus lyophiliser (kept at -20°C). Macroalgae specimen were washed with distilled water after collection and stored at -20°C. Then, the samples were dried using Alpha 1-2 /LD plus lyophiliser and powdered using porcelain mill after freezing with liquid nitrogen. Powdered material was macerated at RT for four hours with 80% ethanol (10 g/100 ml) and filtered through Whatman No. 1 filter paper. The residue from the filter paper was re-suspended in fresh 80% ethanol, re-extracted by maceration for two hours and filtered. Filtrates were combined and evaporated to dryness using rotary evaporator (at 45°C for 30 min) and lyophilisation if necessary. For biological assays, the extracts were dissolved in DMSO to yield stock concentrations of 20-50 mg/ml depending on the assay and stored at -20°C.

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Biological assays

Evaluation of antimicrobial properties

Antimicrobial properties were evaluated against a panel of pathogenic bacteria and fungi as well as fouling bacteria (altogether 20 strains, see Table 1 for details) by using either agar diffusion or broth microdilution assays according to CLSI and EUCAST guidelines [39,40].

For agar diffusion assays, sterile Whatman No. 1 filter paper (GF/C, ø 6 mm) discs were impregnated with the sample (1 mg/disc) and placed on Mueller Hinton agar (MHA) plates which had been previously surface-inoculated with uniformly spread test organism [approximately 1.2 × 108 colony-forming units (CFU)/ml]. Standard antibiotic discs (10 µg/disc) served as positive control (Table 1) and discs containing only solvent as negative control. MHA plates were incubated at 37°C for 24 h (bacteria) or 48 h (fungi) after which the zone of growth-inhibition observed around each disc was measured in millimeters. In broth microdilution assays, inoculum of 5 × 105 CFU/ml in Mueller Hinton broth (MHB) was used for bacteria and 2.5 × 103 CFU/ml in RPMI-1640 (with L-glutamine, w/o NaHCO3 and supplemented with 2% glucose and 0.165 M MOPS, buffered to pH 7) for fungi. After incubation at 37°C for 24 h (bacteria) or at 28°C for 48 h (fungi), absorbance at 620 nm was used for evaluating antimicrobial activity by comparing with controls. Assays were carried out in triplicate.

Evaluation of antiproliferative properties

Cell culture. Two prostate cancer (LNCa, PC-3), a breast cancer (MCF-7) and non-tumorigenic epithelial (MCF-10A) cell lines were used for the evaluation of antiproliferative properties. LNCaP cells were obtained from Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, PC-3 and MCF10A from ATCC (Manassas, VA, USA), and MCF-7 cells from Interlab Cell Line Collection (ICLC, Genova, Italy). LNCaP and PC-3 cells were grown in RPMI-1640, 10% fetal bovine serum, 1% penicillin/streptomycin, and 2mM L-glutamine. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (1 g/l glucose) supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin. MCF-10A were grown in 1:1

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DMEM (4.5 g/l glucose) and HAM F-12, 5% horse serum, 5 µg/ml hydrocortisone, 20 ng/ml epidermal growth factor (EGF), 100ng/ml choleratoxin, 2mM L-glutamine, and 10 µg/ml insulin.

All cells were grown at 37°C/5% CO2.

High throughput anticancer assays. Sample stock solutions were transferred into 384-well master plates and subsequently processed with pipetting robot for 1:10 serial dilutions (20 mg/ml, 2 mg/ml and 0.2 mg/ml) for high-throughput screening. 100 nanolitres of the stock solutions were transferred into white 384-well assay plates (Greiner) in four replicates for ready-made assay plates to be stored in the freezer until used for the screens (final assay concentrations 50, 5 and 0.5 µg/ml). CellTiterGlo (CTG, Promega) proliferation assay was used for assessing anticancer effects of the extracts. DMSO and water were used as negative, and staurosporine and daunorubicine (5 µg/ml, 0.5 µ g/ml, and 0.05 µg/ml of both), as positive controls. Cells (1500 MCF-10A, 1700 PC-3, 2000 MCF-7 and LNCaP cells/well) were plated in 40 µl of culture media into the assay plates on top of the samples. After 72 hours incubation at 37°C/5% CO2, 20 µl of CTG reagent was added, plates were placed in an orbital shaker for 30 minutes for lysis and the luminescence was measured with Envision Multilabel reader (Perkin Elmer). The screens were carried out in two biological replicates in two independent screen sets. The average signals for the four replicates in duplicate screens were calculated and compared to the averages of the control samples (DMSO or water). The screens were technically successful: DMSO controls showed less than 10% CV, the positive controls were very effective and the replicate samples showed very low variation. Due to the fact that the replicate biological screens correlated so well in early screens only one screen was carried out in the later experiments.

Evaluation of anti-inflammatory properties

Primary neuron cell culture preparation and feeding. Timed-pregnant Wistar rats were euthanized by CO2 inhalation according to approved protocols for the care and use of lab animals. Embryonic-day-18 rat whole cortex was used after papain digestion to prepare dissociated cortical cell cultures. Briefly, embryos were removed, chilled on ice, and the cortex was micro-dissected under aseptic conditions. Cortex pieces (1 mm) were digested with papain solution for 30 min at 35°C (carefully inverted every 5 min). The papain solution was removed

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and the cortex pieces were triturated 3 x 1 ml of medium. 20 000 to 50 000 cells per well were plated into 96-well plates in Dulbecco’s modified Eagle’s medium (DMEM) with 10% equine serum (Hyclone) and incubated at 37°C, 5% CO2 for 48-72 hours. Since antibiotics and antimycotics are reported to adversely affect neuron viability, they were not used. Medium was used without glial conditioning, but was stored in a fluorinated ethylene-propylene (FEP) membrane-sealed flask in the incubator to equilibrate the pH and temperature before feeding. No attempt was made to control glial cell proliferation with antimitotic drugs, because glial cells aid in neuronal survival and synaptic development. The cells were fed by completely replacing the media once per week.

Primary astrocyte cell culture preparation and feeding. Two-day old Wistar neonatal rat pups were euthanized in a CO2 chamber. The skin was opened at the midline of the head, from the base of the skull to the mid-eye area, by using micro-dissecting scissors. Skin flaps were folded back, the skull was cut at the midline fissure and the skullcap removed with forceps. The brain was released from the skull cavity along the length of the brain from the olfactory lobes until the beginning of the spinal cord by using a microspatula. The brain was gently transferred to a Petri dish and rinsed with modified DMEM/F12 culture medium supplemented with 10% FBS, 1% glutamine and gentamycin. Each brain was transferred to an inverted Petri dish lid and the cerebrum was first separated from the cerebellum and brain stem, and then the cerebral hemispheres were dissected from each other. A cell suspension was prepared from the cortices by using mechanical digestion. Then, cells were seeded into 75-cm2 tissue culture flasks at a 6 concentration of 15x10 cells and incubated at 37ºC in a humidified atmosphere with 5% CO2 for 48-72 hours.

Primary microglial cell culture preparation, feeding and experiments. Primary microglial cell cultures were prepared from brains of newborn Wistar rats. The neocortex of one-day-old postnatal rats was aseptically dissected out and the meninges and blood vessels were carefully removed. Following cutting in a medium containing 20% horse serum, the suspension was filtered through a 40-mm filter and transferred as a single cell suspension to culture dishes, using

5 mL medium per dish. Cultures were incubated at 37ºC in an atmosphere containing 5% CO2. Minimum Essential Medium Eagle, supplemented with L-glutamine, amino acids, vitamins,

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penicillin, gentamycin, sodium bicarbonate and 20% heat-inactivated horse serum was used as the medium and changed twice a week. After one week, the concentration of horse serum was reduced to 10%. After 16 days in culture, cells were treated with 1µg/ml of lipopolysaccharide (LPS). After six hours, samples were added and plates were incubated for 72 hours at 37ºC, 5%

CO2. Positive controls were treated only with LPS while negative controls were treated with complete medium. After incubation, cells were counted and 2.5 ml of supernatants were collected from each well and frozen at -40ºC for cytokine analyses. All experiments were carried out in duplicate.

MTT reduction assay. For evaluating the effects on neuronal cells and astrocytes, a cell proliferation assay based on MTT was used. Cell lines (1 × 105 cells/ml) were incubated in 96 well plates with extracts for 24 hours. Ten µl of MTT (10 mg/ml) was added to each well and incubated further at 37º C for 4 hours. After incubation, MTT-formazan precipitate was dissolved in 100 µl of DMSO and absorbance was recorded at 570 nm in an automatic plate reader (BioRAD instrument). Data are presented as percentage of survival/activation of treated versus untreated cells.

MHC class-I and MHC class-II antigen expression. For flow cytometric analysis of surface expression of MHC class I (Ox18), MHC class II (Ox6), microglia was prestimulated with LPS (100 ng/ml) and marine extracts for 48 hours. To detach microglia from the surface of the 48-well plastic dishes, they were scraped off with a cell scraper, centrifuged, resuspended in phosphate buffer saline (PBS), and incubated at 4ºC with Ox18 or Ox6 monoclonal antibodies for 30 minutes. After incubation cells were washed twice and resuspended in 500 µl FACS buffer. Flow cytometric analysis was performed by using a FACScan flow cytometer (Becton Dickinson).

Determination of cytokine concentration. The supernatants obtained from microglial cell cultures were analyzed for TNF-α, IL-1β, IL-4, IL-6, IL-8, IL-10, and IL-18 using commercially available ELISA kits following the manufacturers’ instructions. All samples were assayed in duplicate and equivocal results were repeated. The cytokine concentration was calculated from a standard curve of the corresponding recombinant cytokine.

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RESULTS AND DISCUSSION

A set of 98 specimens was collected from 21 collection sites located on the Turkish coast of the Aegean Sea such as Urla, Seferihisar, Urkmez, Burhaniye and Turgutreis (see Table A.1 for details). The set consisted of seven cyanobacteria, 17 microalgae, 72 macroalgae and two seagrass samples, which belonged to seven different phyla; Bacillariophyta (7%), Chlorophyta (27%), Cyanobacteria (7%), Haptophyta (1%), Ochrophyta (40), Rhodophyta (16%) and Tracheophyta (2%). Collected macroalgal/seagrass specimens were washed and freeze-dried prior to extraction. Cyanobacteria and microalgal samples were first purified and then cultivated as monoalgal cultures in laboratory conditions to produce sufficient amounts of biomass for the extraction. Maceration with 80% ethanol was used for preparing crude extracts from the samples, which were then subjected to biological evaluation. The extracts were evaluated by using a panel of 32 assays covering antimicrobial, anti-inflammatory and anticancer targets. By using >50% effect as a threshold, five extracts were identified to be active against pathogenic bacteria and/or fungi, and three against fouling bacteria (Fig. 1). Activity on breast and/or prostate cancer cells were detected for nine extracts and four extracts demonstrated potential activity in the anti- inflammatory evaluation. Interestingly, in most cases the activities were not overlapping between different assay categories. Altogether, 20% of the extracts were defined as potentially active. Majority (17) of the active samples were from macroalgae specimens, and from the phylum Ochrophyta that was also most abundantly represented within the collected set. The following sections describe the found activities in more detail.

Antimicrobial and antifouling activity The inhibitory effects were tested against ten pathogenic strains: three Gram-positive (E. faecalis, S. aureus and methicillin-resistant S. aureus) and four Gram-negative bacteria (E. coli, K. pneumoniae, S. flexneri and V. cholerae), and three fungi (A. fumigatus, C. albicans and C. neoformans). Extracts 10S014, 10S152 and 10S156 showed the most promising results, with growth inhibition above 50% (Fig. 1). Extract of Amphora cf capitellata (10S014) was found to fully inhibit Candida and to inhibit Gram-positive E. faecalis and S. aureus by 68% and 83%, respectively. Interestingly, Nitzschia communis (10S152) fully inhibited the growth of S. aureus at 100 µg/ml concentration. In a similar study conducted by Ördög et al. [41], extracts of

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Chlorella sp. (also studied in this work) were reported to inhibit the growth of several Gram- positive and Gram-negative bacteria at low µg/ml concentrations (MICs between 2-125 µg/ml), but in our study the Chlorella extracts were inactive. These differences may be due to several reasons, such as the difference between the strains [41] as well as in culture conditions and extraction methods. Dussault et al. [9] have reported antimicrobial effects of Padina and Ulva species against foodborne pathogens. However, the reported MIC values against S. aureus are 130 µg/ml, and thus higher than our test concentration (100 µg/ml). Similarly, Mashjoor et al. [42] have reported antimicrobial effects for Ulva and Padina spp., but with MIC values in the range of 93-375 µg/ml.

Cyanobacterial extracts and compounds have previously been reported to possess a variety of biological activities, including antimicrobial properties [43–46]. The cyanobacteria extracts tested in our study did not display significant antimicrobial properties, which is however not surprising considering that our species collection included only seven cyanobacteria species (and not species typically reported to be active, such as Anabaena and Nostoc sp.).

The present study also indicated that two macroalgal species from the Cystoseira genus (10S022- 1 and 10S027) as well as Enteromorpha linza (10S054), exhibited moderate inhibitory activity against fouling marine bacteria (Alcanivorax sp. and Pseudoalteramonas sp.), as shown in Figure 1.

Antiproliferative activity The primary screen results obtained for antiproliferative effects of the extracts are presented in Figure 1. In our study, the extracts of reproductive Dictyopteris membranacea (10S039) and Hypnea musciformis (10S049 and 10S053) displayed a general cytotoxicity against all four cell lines, with inhibition of growth in the range of 60 to 96% with concentration of 50 µg/ml (see Figure 2). However, extracts of species from the Cystoseira genus (10S035, 10S037, 10S038), microalgae Nitzschia thermalis (10S151), and of red algae Laurencia papillosa (10157 and 10S041) displayed cytotoxic effects on the cancer cells (LNCa, PC-3 and MCF-7) but not on the non-tumorigenic cell line MCF-10, suggesting that these extracts might be interesting for further studies regarding their potential anticancer effects. Interestingly, also Hypnea musciformis extract

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10S053 showed this selective antiproliferative effect at the lower test concentration of 5 µg/ml. The extract of brown macroalgae Cystoseira stricta (10S037) showed significant effects on human breast adenocarcinoma MCF-7 cells (inhibition of growth 87%) and similar effect has previously been reported for this species [16]. Recent publications [16,47] have provided evidence that some compounds and pigments present in brown algae are responsible for their antiproliferative activities. All nine Padina pavonica extracts in our study were inactive according to our results. In comparison, Mashjoor et al. [42] showed that brown (Padina antillarum and P. boergeseni) and green (Ulva flexuosa) algae possessed cytotoxic properties against MCF-7 cell line, however, with IC50 values >100 µg/ml.

Anti-inflammatory activity Microglia and astrocytes react to harmful stimuli by producing several inflammatory cytokines such as TNFα, IL-6, IL-1β, and IFN-γ [48]. However, such inflammatory reaction may cause local damage in the tissue and further potentiate inflammation and glial activation, resulting in a persistent inflammatory cycle. This long-term inflammation can give rise to fatal consequences in the CNS, ranging from loss of synapses to impaired cognition and overt neurodegeneration [49]. The results obtained for anti-inflammatory effects of tested extracts are summarized in Figure 1. Extracts from Chaetomorpha aerea (10S018), reproductive Cystoseira crinita (10S038), Dilophus fasciola (10S043) and Caulerpa rasemosa (10S020) clearly showed a reduction in the release of IL-β (50µg/ml: 33%, 29%, 34%, 42%; 100µg/ml: 38%, 35%, 45%, 55%), IL-6 (50µg/ml: 29%, 36%, 35%, 34%; 100µg/ml: 42%, 47%, 45%, 54%), TNF-α (50µg/ml: 32%, 37%, 38%, 42%; 100µg/ml: 44%, 48%, 51%, 55%), and IFN-γ (50µg/ml: 39%, 45%, 47%, 32%; 100µg/ml: 54%, 59%, 65%, 47%), pro-inflammatory cytokines when compared to positive controls. An increase in neuron survival was also observed at both concentrations: 10S018 (50µg/ml: 38%; 100µg/ml: 48%), 10S038 (50µg/ml: 49%; 100µg/ml: 55%), 10S043 (50µg/ml: 53%; 100µg/ml: 63%), and 10S020 (50µg/ml: 43%; 100µg/ml: 48%). The results of both microglial and astrocyte activation indicate a significant activity of 10S018, 10S038, 10S043 and 10S020 extracts (Figure 1), as well as that Dilophus fasciola (10S043) extract exhibit the highest anti-inflammatory activity. Anti-inflammatory properties of two species of cyanobacteria (Chlorella ovalis Butcher and Nannochloropsis oculata Droop), and two microalgae species (Phaeoductylum tricornutum Bohlin and Amphidinium carterae Hulburt) have been studied by

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Samarakoon et al. [50], who demonstrated that N. oculata extracts (at 6.25 µg/ml concentration) show significant activity on nitrogen oxide production in RAW264.7 macrophages. Taken together these results suggest that several marine extracts could be further investigated as therapeutic tools in experimental pathological conditions were microglial and astrocyte activation may contribute to the disease.

CONCLUSIONS

In this article, we presented the results of an extensive study on the bioactivity screening of several species living in the Aegean Sea, i.e., cyanobacteria, micro- and macroalgae. The Aegean Sea, considered as an arm of the Mediterranean Sea (biodiversity hotspot), is an interesting place for the study of marine pharmacology. Based on our results, several macro- and microalgae species showed stimulating results in antimicrobial studies against both pathogenic and fouling microbes. An alga belonging to phylum Rhodophyta, Laurencia papillosa, and three Cystoseira species exhibited promising antiproliferative activity against cancer cell lines. Particularly interesting was the extract from reproductive Cystoseira crinita, which displayed both anti- inflammatory and anticancer effects. Furthermore, an alga belonging to phylum Ochrophyta, Dilophus fasciola, showed significant anti-inflammatory activity. However, further work is required, for example, to evaluate potential side-effects (toxicity) of algal extracts to human body and to fully elucidate the potential of these findings for the development of industrial applications.

Acknowledgements This work was supported by the European Union Seventh Framework Programme FP7 under grant agreement no. 245137 (MAREX) and by the Academy of Finland (grant no. 277001). We want to thank Pirjo Käpylä for the valuable technical assistance in the high throughput anticancer assays carried out at VTT.

Appendix Table A.1. Details on cyanobacteria, micro- and macroalgae specimens collected from the Aegean Sea.

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19

Table 1. Microbial strains used for the evaluation of antimicrobial properties. Strain Positive control Pathogenic bacteria Enterococcus faecalis ATCC 29212a Ciprofloxacin Staphylococcus aureus ATCC 25923a Ciprofloxacin Staphylococcus aureus (MRSA) GMCb Methicillin Escherichia coli ATCC 25922a Ciprofloxacin Klebsiella pneumoniae GMC Streptomycin Shigella flexneri GMC Trimethoprim Vibrio cholerae GMC Chloramphenicol Pathogenic fungi Aspergillus fumigatus GMC Amphotericin B Candida albicans ATCC 90028a Amphotericin B Cryptococcus neoformans GMC Amphotericin B Fouling bacteria Aeromonas hydrophila FCPc Gentamycin Aeromonas salmonicida FCP Gentamycin Alcanivorax borkumensis FCP Gentamycin Alcanivorax sp. FCP Gentamycin Allivibrio salmonicida FCP Gentamycin Erythrobacter litoralis FCP Gentamycin Planococcus donghaensis FCP Penicillin Pseudoalteromonas sp FCP Penicillin Pseudomonas mendocina FCP Penicillin Vibrio furnissii FCP Gentamycin aObtained from Microbiologics, Inc. (USA); bGMC, obtained from Goa Medical College and Hospital; cFCP, fouled copper panels.

20

Figure captions

Figure 1. Antibacterial, antifungal, antifouling, anti-inflammatory and anticancer results for 98 extracts of cyanobacteria, micro- and macroalgae collected from the Aegean Sea. Results are expressed as % effect compared to untreated control. Extracts were tested at final concentration of 100 µg/ml (antimicrobial microdilution assays, anti-inflammation assays), 50 µg/ml (antiproliferation assays) or 1 mg/disc (antimicrobial agar diffusion assays). For clarity, color scale has been applied to highlight the differences in values.

Figure 2. Dose-response results for selected extracts against tumorigenic (MCF-7) and non- tumorigenic (MCF-10) breast cell lines, and two tumorigenic prostate cell lines (LNCaP, PC-3) at 0.5 µg/ml (light grey), 5 µg/ml (mid-grey) and 50 µg/ml (dark grey) concentration.

21 Figure 1.

Antibacterial Antifungal Antifouling Anti-inflammatory Anticancer (MRSA) sp. sp. release release release α γ β

Specimen ID Species faecalis Enterococcus aureus Staphylococcus aureus Staphylococcus coli Escherichia pneumoniae Klebsiella flexneri Shigella cholerae Vibrio fumigatus Aspergillus albicans Candida neoformans Cryptococcus hydrophila Aeromonas salmonicida Aeromonas borkumensis Alcanivorax Alcanivorax salmonicida Allivibrio litoralis Erythrobacter donghaensis Planococcus Pseudoalteromonas Pseudomonas mendocina furnissii Vibrio activation Astrocyte survival Neuronal activation Class I microglial activation Class II microglial IFN- IL-1 release IL-6 TNF- MCF-7 MCF-10 LNCaP PC-3 10S103 Calothrix crustacea 2 -2 0 6 0 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 41 0 2 1 1 2 2 2 4 5 6 2 10S001 Geitlerinema sp. 28 -35 0 2 0 0 0 0 -25 0 0 0 0 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -6 4 10 2 10S003 Halospirulina sp. 4 -15 0 8 0 0 0 0 34 0 0 0 0 0 0 0 0 0 0 0 22 2 3 2 4 1 5 6 -3 -3 4 1 10S004 Oscillatoria rosea -12 -39 0 8 0 0 0 0 23 0 0 0 0 0 0 0 0 0 0 0 25 1 4 3 4 2 2 5 -5 -3 -2 -1 10S155 Oscillatoria sp. -6 -99 0 n.d. 0 0 0 0 23 0 0 0 0 0 0 0 0 0 0 0 26 2 2 1 5 4 3 4 -9 6 11 -5 10S005 Phormidium sp. 9 -4 0 6 0 0 0 0 39 0 0 0 0 0 0 0 0 0 0 0 30 2 5 2 2 1 1 3 0 -5 3 1 Cyanobacteria 10S002 Pseudoscillatoria sp. -1 -11 0 -4 0 0 0 0 21 0 0 0 0 0 0 0 0 0 0 0 24 0 2 3 5 3 1 5 0 1 7 5 10S014 Amphora cf capitellata 68 83 0 15 0 0 0 0 107 0 0 0 0 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -4 8 19 3 10S011 Chlorella sp. -41 -2 0 3 0 0 0 0 -107 0 0 0 0 0 0 0 0 30 0 20 44 0 2 4 5 7 6 4 -6 n.d. 12 2 10S009 Chlorella sp. -77 -20 0 4 0 0 0 0 -170 0 0 0 0 0 0 0 0 0 0 0 36 0 5 4 3 7 6 4 -7 n.d. 10 1 10S159 Chrysoreinhardia sp. -45 -41 0 -1 0 0 0 0 -3 0 0 0 0 0 0 0 0 0 0 0 32 3 4 2 5 4 6 6 2 n.d. 6 4 10S149 Cylindrotheca closterium -6 -16 0 4 0 0 0 0 17 0 0 0 0 0 0 0 0 0 0 0 27 2 2 1 4 0 4 5 -5 8 10 -2 10S008 Dunaliella salina -27 -21 0 4 0 0 0 0 -219 0 0 0 0 0 0 0 0 0 0 0 41 0 4 2 6 5 5 5 -7 n.d. 13 3 10S154 Dunaliella sp. -14 -105 0 n.d. 0 0 0 0 22 0 0 0 0 0 6 0 0 0 0 0 21 1 4 1 4 4 4 5 -4 11 17 0 10S010 Nannochloropsis sp. -48 23 0 7 0 0 0 0 -66 0 0 0 0 0 0 0 0 0 0 0 42 0 3 2 4 6 7 4 -4 n.d. 22 12 10S160 Nanofrustulum sp. -62 -8 0 23 0 0 0 40 -28 0 0 0 0 0 0 0 0 0 0 0 23 2 4 2 8 4 5 6 3 n.d. 8 2 10S152 Nitzschia communis -10 99 0 3 0 0 0 0 -11 0 0 0 0 0 0 0 0 0 0 0 24 1 1 2 3 4 1 5 -7 8 12 0

Microalgae 10S150 Nitzschia sp. -14 -4 0 5 0 0 0 0 33 0 0 0 0 0 0 0 0 0 0 0 23 2 3 2 3 2 5 6 -5 12 14 1 10S151 Nitzschia thermalis -11 -40 0 5 0 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 22 1 2 3 3 4 4 6 38 10 59 72 10S012 Ochrosphaera sp. -9 -71 0 6 0 0 0 0 -222 0 0 0 0 40 0 0 0 33 0 29 39 0 3 2 4 8 8 6 -8 n.d. 14 3 10S153 Phaeodactylum tricornutum 12 -27 0 13 0 0 0 0 -8 0 0 0 0 0 0 0 0 0 0 0 23 2 1 1 3 4 2 4 -7 10 10 0 10S147 Picochlorum sp. -7 -72 0 -2 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 22 5 3 4 4 1 1 5 -6 8 10 -4 10S146 Prasinococcus sp. 4 21 0 10 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 23 4 2 3 2 2 5 6 -5 7 11 -5 10S148 Tetraselmis striata -9 -56 0 -2 0 0 0 0 27 0 0 0 0 0 0 0 13 0 0 0 27 2 1 1 0 0 0 0 -6 10 13 -2 10S099 Caulerpa racemosa -2 0 0 3 0 0 0 0 -28 0 0 6 0 0 0 0 0 0 0 0 35 0 1 1 1 3 2 1 7 7 10 7 10S020 Caulerpa racemosa -40 -22 0 4 0 29 0 0 -41 0 0 0 0 0 0 21 33 0 0 0 54 48 54 45 47 55 39 55 1 n.d. 3 5 10S019 Caulerpa taxifolia 19 -34 0 6 0 0 0 0 -5 0 0 0 0 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -3 8 17 5 10S018 Chaetomorpha aerea -18 -133 0 9 0 0 0 0 30 0 0 0 0 0 0 0 0 0 0 0 59 48 45 45 54 38 42 44 -3 1 1 -6 10S064 Cladostephus hirsutus 0 -47 0 2 0 0 0 0 -34 0 0 0 48 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -2 0 10 -4 10S056 Codium fragile 13 -10 0 1 0 0 0 0 1 0 0 0 37 48 0 0 0 37 0 0 39 0 5 3 5 4 6 4 -2 n.d. 8 0 10S052 Codium fragile 11 3 0 3 0 0 0 0 -73 0 0 0 0 0 0 0 0 0 0 0 35 0 2 2 6 4 4 4 -2 n.d. 10 2 10S040 Codium fragile 3 -38 0 5 0 0 0 0 49 0 0 0 0 0 0 0 0 0 0 0 25 1 3 3 3 4 5 4 -5 7 15 -3 10S032 Colpomenia sinuosa -13 -102 0 15 0 0 0 0 -16 0 0 0 0 0 0 0 0 0 0 0 22 1 7 3 4 6 4 4 -7 4 14 -4 10S045 Colpomenia sinuosa -2 -88 0 0 0 0 0 0 -51 0 0 0 0 0 0 0 0 0 0 0 25 2 3 3 5 1 6 5 -5 5 12 1 10S107 Corallina officinalis 14 -34 0 -24 0 0 0 0 -10 0 0 0 7 0 0 0 0 0 0 0 43 0 2 2 1 7 4 2 6 8 9 6 10S076 Corallina officinalis 9 -76 0 1 0 0 0 0 -46 0 0 0 37 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -3 4 13 1 10S034 Cutleria multifida 9 -52 0 5 0 0 0 0 -42 0 0 0 0 0 0 0 0 0 0 0 21 2 4 4 2 4 4 5 -5 3 12 -4 10S098 Cystoseira barbata -1 -5 0 -4 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 38 0 1 2 2 1 1 1 5 9 9 8 10S065 Cystoseira barbata -39 -46 0 1 0 0 0 0 -137 0 0 0 0 0 0 0 27 40 29 0 27 2 4 4 2 4 1 5 12 n.d. 8 8 10S022 Cystoseira barbata -32 -1 0 0 0 0 0 0 -115 0 0 23 0 0 0 0 0 27 0 0 25 3 3 3 3 5 4 1 4 -4 5 2 10S022-1 Cystoseira barbata 22 -27 0 9 0 0 0 0 -180 0 0 0 44 60 0 0 0 57 0 37 44 0 3 4 5 6 4 4 -3 n.d. 6 2 10S030 Cystoseira barbata -2 -35 0 0 0 0 0 0 -38 0 0 20 0 0 0 0 0 17 0 0 28 2 6 4 3 4 5 5 -5 4 13 -6 10S035 Cystoseira barbata , reproductive 12 -86 0 5 0 0 0 0 -103 0 0 0 0 0 0 0 0 0 0 0 20 4 1 3 3 3 3 3 81 9 39 47 10S044 Cystoseira compressa -33 -11 50 -2 0 0 0 0 -47 0 0 0 0 0 0 32 0 0 0 0 32 2 4 4 4 4 2 2 2 n.d. 3 6 10S110 Cystoseira crinita 9 5 0 4 0 0 0 0 -47 0 0 0 15 0 0 0 33 7 0 0 37 0 1 2 1 1 3 4 2 3 8 2 10S066 Cystoseira crinita 21 12 0 4 0 0 0 0 -46 0 0 0 41 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -1 5 12 0 10S062 Cystoseira crinita 11 -22 0 1 0 0 0 0 -96 0 0 0 44 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -6 7 8 2 10S027 Cystoseira crinita 28 13 0 9 0 0 0 0 -139 0 0 0 48 64 0 0 0 60 0 51 35 0 4 5 5 4 4 2 -1 n.d. 22 8 10S038 Cystoseira crinita, reproductive -1 -44 0 2 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 63 55 49 48 59 35 47 48 89 11 42 59 10S108 Cystoseira spinosa 12 -6 0 -6 0 0 0 0 -1 0 0 6 7 0 0 0 0 0 7 0 57 37 37 39 27 16 23 24 1 7 9 7 10S037 Cystoseira stricta -5 -40 0 -4 0 0 0 0 -45 0 0 0 0 0 0 0 0 0 0 0 22 4 3 1 5 6 6 1 87 -1 41 51 10S058 Dictyopteris membranacea -4 -81 0 11 0 0 0 0 -21 0 0 0 0 0 0 0 0 0 0 0 25 3 3 1 1 5 1 4 2 -7 24 -1 10S039 Dictyopteris membranacea, reproductive -10 -97 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 26 2 2 2 4 4 6 5 95 94 91 70 10S043 Dilophus fasciola 10 -101 0 -2 0 0 0 0 -51 0 0 0 0 0 0 0 0 0 0 0 65 63 55 58 65 45 54 51 -6 1 13 -4 10S109 Enteromorpha compressa 10 -31 0 1 0 0 0 0 5 0 13 6 7 0 0 0 0 0 0 0 38 0 1 2 2 1 2 1 1 5 7 3 10S054 Enteromorpha linza 18 -26 0 -5 0 0 0 0 8 0 0 0 56 56 0 0 0 47 0 37 33 0 3 2 5 5 6 5 -3 n.d. 11 1 10S061 Enteromorpha linza 5 -81 0 1 0 0 0 0 -89 0 0 0 0 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -3 6 10 3 10S028 Enteromorpha linza 25 1 0 -1 0 0 0 0 -147 0 0 0 37 48 0 0 0 47 0 31 36 0 5 4 6 5 3 3 -3 n.d. 8 1 10S017 Enteromorpha linza (Ulva linza) -3 -59 0 0 0 0 0 0 -26 0 0 0 0 0 0 0 0 0 0 0 31 3 4 3 1 1 7 2 0 0 -1 -7 10S048 Gelidium latifolium -50 -33 0 -2 0 0 0 0 -44 0 0 0 0 0 0 26 0 27 0 23 26 3 5 3 3 5 4 3 13 n.d. 9 9 10S024-1 Gracilaria verrucosa 30 -12 0 3 0 0 0 0 -137 0 0 0 0 0 0 0 0 0 0 0 40 0 2 4 4 3 3 1 -1 n.d. 10 3 10S050 Gracilaria verrucosa -1 -17 0 6 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 33 4 4 2 5 5 1 5 -6 6 7 -5 Macroalgae 10S024 Gracilaria verrucosa 24 -16 0 9 0 0 0 0 -147 0 0 0 48 44 0 0 0 47 0 37 41 0 5 5 5 4 3 2 -4 n.d. 6 1 10S047 Gracilaria verrucosa, carposporophyte -10 -97 0 19 0 0 0 0 -7 0 0 0 0 8 0 0 0 0 0 0 29 1 2 2 4 2 2 4 -6 3 10 -4 10S105 Halopteris scoparia 1 46 0 -19 0 0 0 0 -11 0 0 0 7 24 0 0 0 0 0 0 41 0 1 2 2 2 4 2 3 3 7 3 10S068 Halopteris scoparia -3 -104 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0 2 11 -2 10S075 Halopteris scoparia 14 -73 0 5 0 0 0 0 9 0 0 0 37 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -6 4 14 3 10S029 Halopteris scoparia 18 -22 0 0 0 0 0 0 -232 0 0 0 0 0 0 0 0 0 0 0 37 0 4 3 7 4 4 4 -3 n.d. 9 2 10S049 Hypnea musciformis -5 -30 0 2 0 0 0 0 -26 0 0 0 0 40 0 0 0 0 0 0 32 5 3 3 1 1 1 6 96 95 92 65 10S053 Hypnea musciformis -4 -95 0 -2 0 0 0 0 -51 0 0 0 0 0 0 0 0 0 0 0 23 6 2 1 0 6 0 3 96 96 94 60 10S033 Jania rubens 1 -30 0 -3 0 0 0 0 -238 0 0 0 0 0 0 0 0 0 0 0 65 45 53 55 28 25 27 22 -1 n.d. 10 3 10S125 Laurencia obtusa -54 -44 0 -6 0 0 0 0 -75 0 0 0 0 0 0 0 0 33 0 0 21 1 3 3 1 3 5 6 30 n.d. 30 6 10S063 Laurencia papillosa 5 -21 0 2 0 0 0 0 -54 0 0 0 0 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -8 5 6 -3 10S157 Laurencia papillosa -52 -41 0 0 0 0 0 0 -133 0 0 0 0 0 0 0 0 0 0 0 24 3 4 2 4 4 5 1 55 n.d. 44 14 10S041 Laurencia papillosa 10 17 0 14 0 0 0 0 -56 0 0 0 0 0 0 0 0 0 0 0 30 2 3 2 5 1 4 6 91 28 56 71 10S073 Liagora viscida -6 -26 0 2 0 0 0 0 -30 0 0 0 0 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -4 9 11 -1 10S102 Padina pavonica 11 -20 0 -11 0 0 0 0 -11 0 0 0 7 0 0 0 20 0 11 0 50 36 39 44 27 23 26 27 3 5 10 7 10S104 Padina pavonica -1 0 0 1 0 0 0 0 -9 0 10 6 0 0 0 0 7 0 0 0 42 0 2 2 3 3 3 3 3 6 8 4 10S100 Padina pavonica 19 -33 0 -13 0 0 0 0 -5 0 0 0 0 0 0 0 0 0 0 0 39 0 1 1 1 4 3 2 0 5 10 4 10S067 Padina pavonica 17 -21 0 2 0 0 0 0 -39 0 0 0 33 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0 4 11 1 10S074 Padina pavonica -3 -66 0 3 0 0 0 0 -53 0 0 0 44 12 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -5 2 7 -4 10S059 Padina pavonica 16 26 0 2 0 0 0 0 -50 0 0 0 0 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -2 8 12 1 10S023 Padina pavonica -16 -60 0 24 0 0 0 0 5 0 0 0 0 0 0 0 0 13 0 0 22 1 3 3 4 5 6 5 -1 2 7 -3 10S023-1 Padina pavonica 39 7 0 9 0 0 0 0 -209 0 0 0 44 48 0 0 0 40 0 29 42 0 6 2 4 3 5 6 -6 n.d. 12 3 10S072 Padina pavonica -1 -75 0 2 0 0 0 0 -55 0 0 0 44 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -4 4 7 -1 10S021 Petalonia fascia -37 -17 50 1 0 0 0 0 -51 0 0 0 0 0 0 21 0 0 0 0 23 3 4 4 5 6 2 2 1 0 4 3 10S156 Polysiphonia elongata -55 -2 70 7 0 43 0 40 -34 0 0 0 0 0 0 21 0 33 0 0 23 2 5 3 5 5 2 4 9 n.d. 5 6 10S042 Sargassum vulgare 9 -16 0 4 0 0 0 0 -35 0 0 0 0 0 0 0 0 0 0 0 28 1 4 4 4 4 5 5 45 8 16 14 10S106 Taonia atomaria 1 -30 0 -5 0 7 0 0 -3 0 0 0 7 0 0 0 7 0 0 0 42 0 2 1 1 4 3 3 3 5 8 5 10S101 Ulva rigida 0 -23 0 -13 0 0 0 0 -7 0 0 0 0 0 0 0 0 0 0 0 37 0 1 1 2 6 2 2 4 2 9 4 10S016 Ulva rigida -10 -88 0 -1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 32 0 3 2 4 5 0 3 -1 0 -1 -5 10S016-1 Ulva rigida 13 -29 0 1 0 0 0 0 -282 0 0 0 41 0 0 0 0 37 0 29 45 0 2 2 6 4 5 6 -7 n.d. 10 1 10S051 Ulva rigida 5 11 0 10 0 0 0 0 -35 0 0 0 0 0 0 0 0 0 0 0 36 3 3 1 6 4 5 4 -5 6 10 -5 10S060 Ulva rigida 5 -68 0 8 0 0 0 0 -92 0 0 0 0 0 0 0 0 0 0 0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. -6 6 13 1 10S015 Ulva rigida -4 -66 0 2 0 0 0 0 -7 0 0 0 0 0 0 0 0 0 0 0 33 1 6 3 3 2 7 4 3 0 3 2 10S025 Ulva rigida -13 -42 0 -5 0 0 0 0 -41 0 0 0 0 0 0 0 0 0 0 0 25 2 4 4 3 4 6 4 -3 -2 3 -4 10S026 Posidonia oceanica 10 -58 0 4 0 0 0 0 -5 0 0 0 0 0 0 0 0 0 0 0 27 1 3 5 4 5 4 6 -4 5 15 -3 Sea grass 10S036 Zostera marina 9 -28 0 3 0 0 0 0 7 0 0 0 0 0 0 0 0 0 0 0 19 2 2 2 3 1 4 4 -6 5 11 -3

n.d., not determined 22 -100 -50 0 50 100

Figure 2.

100 100 100 10S035 10S037 10S038 80 80 80

60 60 60

40 40 40

20 20 20 Inhibition of growth (%) Inhibition of growth 0 0 0 MCF-7 MCF-10 LNCaP PC-3 MCF-7 MCF-10 LNCaP PC-3 MCF-7 MCF-10 LNCaP PC-3

100 100 100 10S039 10S041 10S049 80 80 80

60 60 60

40 40 40

20 20 20 Inhibition of growth (%) Inhibition of growth 0 0 0 MCF-7 MCF-10 LNCaP PC-3 MCF-7 MCF-10 LNCaP PC-3 MCF-7 MCF-10 LNCaP PC-3

100 100 100 10S151 10S053 10S157 80 80 80

60 60 60

40 40 40

20 20 20 Inhibition of growth (%) Inhibition of growth 0 0 0 MCF-7 MCF-10 LNCaP PC-3 MCF-7 MCF-10 LNCaP PC-3 MCF-7 MCF-10 LNCaP PC-3

23