Bioactivity of marine organisms: Part VI--Screening of some marine flora from Indian coasts.

Alcoholic extracts of 50 botanically identified species of marine flora have been screened for a wide range of biological activities. Of these, 2 extracts exhibited anti-amoebic and antiviral activity each, 3 of them had anti-implantation activity; 9 had hypoglycaemic activity while hypotensive activity was associated with 11 extracts; 14 extracts were found to be diuretic and 1 of them had anti-inflammatory activity. Further, 10 of these extracts exhibited 2 types of activities while a combination of 3 and 4 types of activities was observed in one extract each. Follow-up studies have been carried out in some plants with confirmed activity. The active principles and results of these studies are reported.     

Bioactivity of marine organisms: Part IX--screening of some marine flora from the Indian coasts

Alcoholic extracts of 48 identified species of marine flora were screened for a wide range of biological activities. Of these, 3 extracts showed diuretic activity while 2 extracts showed hypotensive effect.     

Antibacterial activity in four marine crustacean decapods

A search for antibacterial activity in different body-parts of Pandalus borealis (northern shrimp), Pagurus bernhardus (hermit crab), Hyas araneus (spider crab) and Paralithodes camtschatica (king crab) was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid, and further extracted and concentrated on C18 cartridges. Eluates from the solid phase extraction were tested for antibacterial, lysozyme and haemolytic activity. Antibacterial activity against Escherichia coli, Vibrio anguillarum, Corynebacterium glutamicum and Staphylococcus aureus was detected in extracts from several tissues in all species tested, but mainly in the haemolymph and haemocyte extracts. V. anguillarum and C. glutamicum were generally the most sensitive micro-organisms. In P. borealis and P. bernhardus most of the active fractions were not affected by proteinase K treatment, while in H. araneus and P. camtschatica most fractions were sensitive to proteinase K treatment, indicating antibacterial factors of proteinaceous nature. In P. bernhardus the active fractions were generally heat labile, whereas in H. araneus the activities were resistant to heat. Differences between active extracts regarding hydrophobicity and sensitivity for heat and proteinase K treatment indicate that several compounds are responsible for the antibacterial activities detected. Lysozyme-like activity could be detected in some fractions and haemolytic activity against human red blood cells could be detected in haemolymph/haemocyte and exoskeleton extracts from all species tested.     

Screening of the Antarctic marine sponges (Porifera) as a source of bioactive compounds

Sponges (Porifera) currently represent one of the richest sources of natural products and account for almost half of the pharmacologically active compounds of marine origin. However, to date very little is known about the pharmacological potential of the sponges from polar regions. In this work we report on screening of ethanolic extracts from 24 Antarctic marine sponges for different biological activities. The extracts were tested for cytotoxic effects against normal and transformed cell lines, red blood cells, and algae, for modulation of the activities of selected physiologically important enzymes (acetylcholinesterase, butyrylcholinesterase, and α-amylase), and for inhibition of growth of pathogenic and ecologically relevant bacteria and fungi. An extract from Tedania (Tedaniopsis) oxeata was selectively cytotoxic against the cancer cell lines and showed growth inhibition of all of the tested ecologically relevant and potentially pathogenic fungal isolates. The sponge extracts from Isodictya erinacea and Kirkpatrickia variolosa inhibited the activities of the cholinesterase enzymes, while the sponge extracts from Isodictya lankesteri and Inflatella belli reduced the activity of α-amylase. Several sponge extracts inhibited the growth of multiresistant pathogenic bacterial isolates of different origins, including extended-spectrum beta-lactamase and carbapenem-resistant strains, while sponge extracts from K. variolosa and Myxilla (Myxilla) mollis were active against a human methicillin-resistant Staphylococcus aureus strain. We conclude that Antarctic marine sponges represent a valuable source of biologically active compounds with pharmacological potential.     

Screening and preliminary characterization of hemagglutinins in Vietnamese marine algae

Extracts from 44 species of Vietnamese marine algae, including 15 Chlorophyta, 18 Rhodophyta and 11 Phaeophyta species, were examined for hemagglutination activity with a variety of different animal and human erythrocytes that were untreated or treated with enzymes. Almost all extracts showed activity toward at least one type of erythrocytes, although those from three Chlorophyta and two Rhodophyta species showed no hemagglutination with any type of erythrocytes examined. Strong activity was detected in extracts from two Chlorophyta (Anadyomene plicata and Avrainvillea erecta) and four Rhodophyta species (Gracilaria eucheumatoides, Gracilaria salicornia, Kappaphycus alvarezii, and Kappaphycus striatum) with enzyme-treated rabbit and sheep erythrocytes. The hemagglutinins of seven Chlorophyta and eight Rhodophyta species were examined for sugar-binding specificity, pH- and temperature-stability, and divalent cation-independency of hemagglutination using ammonium sulfate-precipitates prepared from their extracts. In a hemagglutination-inhibition test with various monosaccharides and glycoproteins, none of the hemagglutinins had affinity for monosaccharides, except the Codium arabicum and Gracilaria euchematoides hemagglutinins, whose activities were inhibited by both N-acetyl-d-galactosamine and N-acetyl-d-glucosamine. On the other hand, all of the hemagglutinins activities were inhibited by some glycoproteins. The inhibition profiles with glycoproteins were different depending on hemagglutinin species, and suggest the presence of lectins specific for high mannose N-glycans, complex N-glycans, or O-glycans. The activities of these algal hemagglutinins were stable over a wide range of pH and temperature, and independent of the presence of divalent cations. These results indicate that Vietnamese marine algae are a good source of novel and useful lectins.     

Antifouling activities of octocorals on some marine microfoulers

The antifouling activities of vacuum-dried 70% aqueous alcohol extracts of four gorgonian and five soft corals against four dominant marine fouling diatoms (Navicula subinflata Grun, N. crucicula Smith, Amphora sp., Nitzschia sp.) are described. Of the 36 possible combinations (9 corals x 4 diatoms) 23 of the interactions (64%) showed 100% activity. Extracts of the gorgonian coral Echinogorgia complexa Nutting and the soft coral Dendronephthya (Morchellana) sp. showed 100% growth inhibition against all four fouling diatoms, implying the presence of potent broad spectrum antifouling compounds; other extracts showed more limited species specificity. Exposed cells when transferred to extract-free media, resumed normal growth indicating a nontoxic way of action. The effective concentration for 50% growth inhibition of the attached cells (EC50) varied for each extract and test organism used. The EC50 values for the extract of the gorgonian coral E. complexa against the fouling diatoms ranged from 86 microg/ml (N. subinflata) to 505 microg/ml (Amphora sp.) whereas the EC50 values for extracts of the soft coral Dendronephthya (Morchellana) sp. varied from 28 microg/ml (N. crucicula) to 415 microg/ml (Amphora sp.). The results support the hypothesis that octocorals contain antifouling agents, which could be exploited for the development of nontoxic natural antifouling technology.     

Hydrocarbon metabolism in a marine bacterium]

The marine bacterium L.16.1 (Alcaligenes sp.) grows preferentially on alkanes (C10 to C18) with a very high growth yield (98 per cent); optimal growth depends strictly on the presence of a well-defined NaCl concentration (100 mM). Our strain is constitutive for the enzymatic systems responsible for the oxidation of alkanes to fatty acids, i.e. NADH-dependent hydroxylase, alcohol and aldehyde dehydrogenases, the latter of which located at the cytoplasmic membrane level. The aerobic oxidation of primary alcohols by particulate extracts prepared in the presence of 400 mM NaCl is NAD+-dependent (Km = 0.082 mM, Vmax = 238 with decanol). With extracts prepared in the absence of NaCl, Vmax undergoes a very strong decrease. On the contrary , the NAD+ (P)+-dependent oxidation of aldehydes is carried out anaerobically by the same extracts irrespective of the presence or the absence of added Na+ in the solutions used for the preparation of these extracts. A possible explanation for our results could be that Na+ acts on the enzymatic systems for which the maintenance of the membrane integrity is essential. This interpretation is consistent with the slowing down of the growth speed accompanying the decrease of NaCl concentration in the growth medium. With regard to alcohol and aldehyde-dehydrogenases, it is noteworthy that these enzymes behave like similar enzymatic activities induced by alkanes in other microorganisms.     

Differential growth inhibition of cancer cell lines and antioxidant activity of extracts of red, brown, and green marine algae

As the use of various anticancer drugs is associated with many undesirable side effects, there is an urgent need for the discovery of new, better, and specific anticancer compounds. Antioxidant and antiproliferative activities as well as effects on cell morphology were investigated for methanol (M), chloroform (C), ethyl acetate (E), and aqueous (A) extracts of Caulerpa peltata, Gelidiella acerosa, Padina gymnospora, and Sargassum wightii using 2,2-diphenyl-1-picrylhydrazyl radical-scavenging, ferrous ion chelation, and resazurin-based growth inhibition (in A549, HCT-15, MG-63, and PC-3 cell lines) assays. A general trend was the greater extraction of phenols and flavonoids by chloroform and ethyl acetate, which showed higher activity in many assays. These non-polar C and E extracts showed higher DPPH radical-scavenging and growth inhibitory activities in A549, HCT-15, and PC-3 cells. However, higher ferrous ion chelation (A extracts) and growth inhibition in MG-63 cells (M and A extracts) were seen for the polar extracts. Furthermore, P. gymnospora and C. peltata emerged as promising sources for antiproliferative agents that could be explored for their own activity and as leads for the development of other compounds.     

Antibacterial activity of marine macroalgae against fish pathogenic Vibrio species

In mariculture, diseases of microbial origin can cause significant economic losses worldwide; the evolution of microorganism resistance to antibiotics has resulted in a growing need for new antibacterial compounds that are effective in veterinary medicine and characterized by limited undesirable side effects. Increased attention has recently been turned to seaweeds as a promising source for metabolites with antimicrobial activity. Vibriosis is a common disease, caused by bacteria of the genus Vibrio, that can result in high mortality in aquaculture. The aim of this study was to identify seaweeds with antibacterial activity against some pathogenic Vibrio species, in order to identify a possible alternative to the commonly used antibiotics in aquaculture. Chloroform/methanol lipidic extracts of six seaweed species (Chaetomorpha linum, Cladophora rupestris, Gracilaria dura, Gracilaria gracilis, Gracilariopsis longissima, Ulva prolifera) were tested for their antibacterial activities against six fish pathogenic Vibrio species using the disc diffusion method. Different susceptibilities to lipidic algal extracts were observed. All six of the seaweed extracts tested demonstrated inhibition of Vibrio ordalii. The best was that from Gracilariopsis longissima, showing activity against Vibrio ordalii, Vibrio salmonicida, Vibrio alginolyticus and Vibrio vulnificus. The results confirmed the potential use of seaweed extracts as a source of antibacterial compounds or as a health-promoting feed for aquaculture.     

Thymidine phosphorylating enzymes in the gonads of marine invertebrates

The activities of enzymes involved in the consecutive phosphorylation of thymidine were revealed in the gonad extracts of marine invertebrates. Along with thymidine kinase activity, thymidilate kinase activity was revealed in all the studied species; however, the specific activities of nucleoside and nucleotide kinases varied in different species of mollusks, sea stars and sea urchins. Thymidine and thymidilate kinases were isolated from the gonads of the scallop Mizuhopecten yessoensis and some of their enzymat properties were studied. The thymidine kinase of M. yessoensis catalyzed the phosphorylation of thymidine and deoxycytidine at a lesser rate, but didn’s use purine ribo-and deoxyribonucleosides or pyrimidine ribonucleosides as phosphate acceptors. The thymidilate kinase carried out both TMP and dCMP phosphorylation. As well as ATP, the enzymes of M. yessoensis were also able to use dATP, dGTP, GTP, UTP and CTP as donors of phosphate groups. The thymidine kinase activity was inhibited by TMP, TTP and dCTP.     

Antibacterial activities of anthozoan corals on some marine microfoulers

The antibacterial activities of twelve species of anthozoans (4 gorgonians, 5 soft corals and 3 antipatharians) collected off the east coast of India were assayed against four dominant marine fouling bacterial strains isolated from the biofilm of fouled aluminium panels. Of the 48 combinations (12 corals x 4 bacteria) eighteen interactions showed antibacterial activity (37.5%). Such activity was most apparent in gorgonians, which inhibited bacterial growth in ten out of sixteen interactions (62.5%) compared with that of five out of twenty interactions (25%) among soft corals and three out of twelve interactions (25%) among antipatharians. The activity scores varied with different extracts and test organisms used, and was highest in antipatharians. Among the four bacterial strains Vibrio sp. was the least sensitive (2/12) when compared with Flavobacterium sp. (6/12). This is the first report of antibacterial activities of antipatharian colonies against marine microfoulers. The results imply that anthozoan corals harbour potent agents which could be exploited for the development of antifouling technology.     

A new screening for hemagglutinins from Vietnamese marine macroalgae

Aqueous extracts from 42 species of Vietnamese marine macroalgae, including 17 Chlorophyta, 22 Rhodophyta, and three Phaeophyta species, were examined for hemagglutination activity using native and enzyme-treated different animal and human erythrocytes. All extracts agglutinated at least one type of erythrocytes tested. Strong activity was detected in extracts from four Chlorophyta (Caulerpa serulata var. boryana, Caulerpa sertularioides f. longipes, Halimeda velasquezii, and Halimeda discoidea) and two Rhodophyta species (Gelidiella acerosa and Titanophora pulchra) with enzyme-treated rabbit and horse erythrocytes. The hemagglutinins of some active species were examined for sugar-binding specificity, pH, temperature stability, and divalent cation independency using ammonium sulfate precipitates prepared from their extracts. In a hemagglutination–inhibition test with various monosaccharides and glycoproteins, none of the hemagglutinins had affinity for monosaccharides. The activity of the hemagglutinins was inhibited by some glycoproteins tested. The inhibition profiles with glycoproteins were different depending on hemagglutinin species, suggesting the presence of lectins specific for complex N-glycans, high mannose N-glycans or O-glycans. On the other hand, the activities of almost all algal hemagglutinins were stable over a wide range of pH and temperature, and independent of the presence of divalent cations, except Gelidiopsis scoparia hemagglutinin, its activity was dependent on the presence of divalent cations. These results suggest that Vietnamese marine macroalgae may be good sources of useful lectins for many biological applications.     

Simple separation of anticoagulant sulfated galactan from marine red algae

In this study, hot water extracts of 22 red algal species were evaluated for their potential anticoagulant activities. The extracts from eight species (Grateloupia elliptica, Sinkoraena lancifolia, Halymenia dilatata, Grateloupia lanceolata, Lomentaria catenata, Martensia denticulata, Schizymenia dubyi, Chondrus crispus) showed potent activated partial thromboplastin time (APTT). Of these eight algae, the crude polysaccharide fraction (CpoF) from the hot water extracts of L. catenata and S. dubyi showed the highest APTT activity. Lomentaria catenata and S. dubyi were selected and an enzymatic digestion process which could effectively separate a crude polysaccharide fraction with higher yields from raw algae materials was applied. The 10 enzymes tested included five carbohydrases and five proteases. The Ultraflo and Celluclast digests of L. catenata and the AMG digest of S. dubyi exhibited the most potent anticoagulant activity. Furthermore, in both species, the active compounds were mainly concentrated in the >30 kDa fraction through ultrafiltration and showed strong APTT (>1000) and thrombin time (TT) >1000 activity. The active compounds were shown to be sulfated galactans with a greater than 80% galactose content and an 0.22 ∼ 0.31 sulfate to total sugar ratio.     

Microassays for a set of enzymes in individual small marine copepods

Fluorogenic assays for a set of five hydrolytic enzymes involved in digestion and food utilization (alanine and arginine aminopeptidase, lipase/esterase, chitobiase, and beta-glucosidase) were optimized to measure activities of these enzymes in the same extracts of individual small North Sea copepods. The enzyme activities of Acartia clausi, Centropages typicus, Corycaeus anglicus, Paracalanus parvus, and Temora longicornis showed distinct species specific activity patterns, but also high intra-specific variability. Protein, lipids, carbon and nitrogen (C, N) were determined with micro-scale assays in individual copepods or in batches of 10 to 50 animals. Water soluble protein contents ranged from 16% to 38%, and lipid contents from 2.4% to 5.5% of dry mass. The molar C/N ratios were between 4.1 and 4.5. The presented microassays provide suitable tools for studying physiological reactions of copepods and other small pelagic crustaceans in response to variable environmental conditions.     

The marine red alga Eucheuma serra J. Agardh, a high yielding source of two isolectins

Aqueous ethanolic extracts from five species of the genus Eucheuma (Rhodophyta) i.e. E. serra, E. amakusaensis, E. cottonii, E. gelatinae and E. denticulatum, were examined for hemagglutinating activity with vertebrate erythrocytes. All the extracts tested agglutinated trypsin-treated sheep and rabbit erythrocytes as well as untreated sheep erythrocytes. From the extract of E. serra, which exhibited the highest activity, a lectin was purified by precipitation with cold ethanol followed by gel filtration to exhibit a single band on SDS-PAGE. The yield was surprisingly as high as 1000 mg from 100 g powdered alga. The purified lectin was further separated into two isoforms, designated ESA-1(90 mg) and ESA-2 (890 mg), by ion exchange chromatography. Both lectins showed a single protein band with the same molecular weight of 29 000 on SDS-PAGE and differed from each other only in isoelectric point (pI 4.75 for ESA-1 and pI 4.95 for ESA-2). Biochemical studies revealed that both are monomeric proteins without a carbohydrate moiety. The hemagglutinating activities were stable over a wide pH range and at a relatively high temperature. The activities were inhibited by a number of glycoproteins, but not by any of the monosaccharides and disaccharides tested. The lectins showed strong mitogenic activities for mouse lymphocytes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antibiotic activity of lectins from marine algae against marine vibrios

Saline and aqueous ethanol extracts of marine algae and the lectins from two red algal species were assayed for their antibiotic activity against marine vibrios. Experimental studies were also carried out on the influence of environmental factors on such activity, using batch cultures. The results indicated that many of the saline extracts of the algal species were active and that the activity was selective against those vibrios assayed. The algal extracts were active against Vibrio pelagius and the fish pathogen V. vulnificus, but inactive against V. neresis. Algal lectins from Eucheuma serra (ESA) and Galaxaura marginata (GMA) strongly inhibited V. vulnificus but were inactive against the other two vibrios. The antibacterial activity of algal extracts was inhibited by pretreatment with various sugars and glycoprotein. Extracts of the two red algae, E. serra and Pterocladia capillacea, in saline and aqueous ethanol, inhibited markedly the growth rate of V. vulnificus at very low concentrations. Culture results indicated that metabolites active against V. vulnificus were invariably produced in P. capillacea over a wide range of temperature, light intensity, and nutritional conditions. Enhanced antibacterial activity occurred when P. capillacea was grown under higher irradiance, severe nutrient stress and moderate temperature (20 °C), reflecting the specific antibiotic characteristics of this alga. The strong antibiotic activity of lectins towards fish pathogenic bacteria reveals one of the important roles played by algal lectins, as well as the potential high economic value of those marine algae assayed for aquaculture and for biomedical purposes.     

Neurotoxic activity induced by a haemolytic substance in the extract of the marine sponge Geodia corticostylifera

In our search for marine bioactive compounds we chose a Brazilian Coast sponge, Geodia corticostylifera (Demospongiae), whose extracts showed previously antibacterial and antifungal activities. In the present work we studied the following toxic properties of G. corticostylifera extract: neurotoxic (in mouse neuromuscular junction); mouse acute toxicity (IP) and haemolytic (against mouse and frog erythrocytes). Insertion of ionic channels in planar lipid bilayers in presence of a haemolytic purified fraction of the extract was observed. The toxic activities of G. corticostylifera crude extract are related to the formation of ionic pores in the cell membrane, which induce the release of haemoglobin from erythrocytes, and depolarization of nerve and muscle membranes. These last physiological effects cause the blockade of the diaphragm contractions, leading to death through respiratory arrest.     

Endogenous opioids in marine invertebrates

A study of the presence of opioids in invertebrates was carried out on 11 species of marine organisms, ranging from sponges to tunicates. Delipidized acid-acetone extracts from whole organisms or dissected organs were assayed by receptor binding assays and radioimmuno assays. The extracts from all species tested were found to contain substances capable of competing in both assays with opioid peptides.     

A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products

Background  

The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity.     

Demonstration of HOQNO and antimycin A sensitive coupling of NADH oxidation and APS and sulfite reduction in a marine Desulfovibrio strain

Abstract In cell suspensions of the marine sulfate-reducing bacterium Desulfovibrio 20020 (DSM 3099) permeabilized with formaldehyde or Triton X-100, sulfite-dependent NADH oxidation activities of 0.05 μmol · min⁻¹· mg⁻¹ protein were detected. NADH oxidation coupled to APS, thiosulfate and fumarate reduction was also demonstrated. All the activities were subject to inhibition by HOQNO and antimycin A. The rate of NADH oxidation coupled to the reduction of sulfite was extremely low in cell-free extracts. The physiological function and possible mechanism of the NADH oxidation coupled to the reduction of various electron acceptors are discussed.