Investigations into the Toxicology of Spirolides, a Group of Marine Phycotoxins

Spirolides are marine phycotoxins produced by the dinoflagellates Alexandrium ostenfeldii and A. peruvianum. Here we report that 13-desmethyl spirolide C shows little cytotoxicity when incubated with various cultured mammalian cell lines. When administered to mice by intraperitoneal (ip) injection, however, this substance was highly toxic, with an LD50 value of 6.9 µg/kg body weight (BW), showing that such in vitro cytotoxicity tests are not appropriate for predicting the in vivo toxicity of this toxin. Four other spirolides, A, B, C, and 20-methyl spirolide G, were also toxic to mice by ip injection, with LD50 values of 37, 99, 8.0 and 8.0 µg/kg BW respectively. However, the acute toxicities of these compounds were lower by at least an order of magnitude when administration by gavage and their toxic effects were further diminished when administered with food. These results have implications for future studies of the toxicology of these marine toxins and the risk assessment of human exposure

Bioactive triterpenoids of the Rutales.

This thesis describes the investigation of the bioactivity of triterpenoids, limonoids and spiro-triterpenoids, isolated from the order of the Rutales. The focus of this research has been towards the development of botanical insecticides with non-neurotoxic modes of action in insects. It follows the recent development of Neem (Azadirachta indica, Meliaceae) as a botanical insecticide containing a limonoid azadirachtin as its main active compound. Because so few Meliaceae members have been examined for insecticidal activity the project was initiated by the screening of over sixty ethanol extracts of various parts of twenty species (collected from Costa Rica and the Tropical Museum in Miami, Florida) for bioactivity against the European corn borer (Ostrinia nubilalis) and the Variegated cutworm (Peridroma saucia). The data suggest that a majority of the extracts studied inhibited growth significantly; some are more active than neem leaf extracts. Swietenia mahogani bark, Trichilia glabra bark, T. hirta leaves, T. americana bark, T. trifolia wood, T. pleana and Azadirachta indica wood showed potent activity against the cutworm. Ruptiliocarpon caracolito bark, Cedrela odorata leaves, Aphanamixis polystachys wood, T. glabra wood and T. pleana bark showed good activity against the corn borer larvae. Bioassay guided fractionation of the most active crude extract, the bark of Ruptiliocarpon caracolito, has resulted in the isolation of six very novel spiro-CD-triterpenoids, the spirocaracolitones. The spirocaracolitones, at 100 ppm, were screened for antifungal activity against Fusarium using a hyphal growth bioassay. After 48 h a high degree of inhibition of growth (59-79%) was observed. These novel triterpenoids exhibited no antimalarial activity when screened against Plasmodium falciparum. A study of the effects of these compounds, incorporated into artificial diet, on the neonate life cycle of the European corn borer was conducted. All the spirocaracolitones effected the growth and the development of the insects but spirocaracolitone B and spirocaracolitone C were substancially more active than the other spirocaracolitones. Two postulates dealing with the biosynthesis of these unique spiro-compounds are also presented. Both commence with a friedelin derivative because canophyllol was isolated in large quantities from the hexane extract. They envisage generation of a carbocation at C12 followed by migration of the C23 methyl group this results in the formation of a carbocation at C13. The spirosystem results from migration of the C8-C14 bond. An investigation of the structure/activity relationships of gedunin was conducted to determine the moieties responsible for its antimalarial and/or antifeedant properties. Ten derivatives of gedunin were prepared (1,2-dihydrogedunin, 1,2-epoxygedunin, 1,2-dihydro-3$\beta$-gedunol, 3$\beta$-acetoxy-1,2-dihydrogedunin, 7-deacetylgedunin, 7-ketogdunin, hexahydrogedunin, tetrahydrogedunin, 21-acetylgedunin, 23-acetylgedunin) along with five other limonoids (limonin, epilimonol, nomilin, obacunone, hirtin) closely related to gedunin were evaluated for antifeedant activity (Ostrinia nubilalis) and antimalarial activity (Plasmodium falciparum). Evaluation of these compounds for antimalarial activity presented no increase in activity however it was clearly determined that alterations to the enone in ring A and at C7 of gedunin resulted in large losses of activity. Evaluation of the effects of these limonoids on the neonate life cycle of the European corn borer at 5 and 50 ppm incorporation into the artificial diet did lot produced dramatic effects on the growth and development of the larvae. At these concentrations structure/activity relationships could not be proposed.\sp* ftn\sp*Please refer to the dissertation for diagrams

A comparative study on innate immune parameters in the epidermal mucus of various fish species.

Peer reviewed: YesNRC publication: Ye

Myxinidin, a Novel Antimicrobial Peptide from the Epidermal Mucus of Hagfish, Myxine glutinosa L.

Fish epidermal mucus contains innate immune components that provide a first line of defense against various infectious pathogens. This study reports the bioassay-guided fractionation and characterization of a novel antimicrobial peptide, myxinidin, from the acidic epidermal mucus extract of hagfish (Myxine glutinosa L.). Edman sequencing and mass spectrometry revealed that myxinidin consists of 12 amino acids and has a molecular mass of 1,327.68 Da. Myxinidin showed activity against a broad range of bacteria and yeast pathogens at minimum bactericidal concentration (MBC) ranging from 1.0 to 10.0 \ub5g/mL. Screened pathogens, Salmonella enterica serovar Typhimurium C610, Escherichia coli D31, Aeromonas salmonicida A449, Yersinia ruckeri 96-4, and Listonella anguillarum 02-11 were found to be highly sensitive to myxinidin at the MBC of 1.0\u20132.5 \ub5g/mL; Staphylococcus epidermis C621 and yeast (Candida albicans C627) had an MBC of 10.0 \ub5g/mL. The antimicrobial activity of myxinidin was found to be two to 16 times more active than a potent fish-derived antimicrobial peptide, pleurocidin (NRC-17), against most of the screened pathogens. The microbicidal activity of myxinidin was retained in the presence of sodium chloride (NaCl) at concentrations up to 0.3 M and had no hemolytic activity against mammalian red blood cells. These results suggest that myxinidin may have potential applications in fish and human therapeutics.Peer reviewed: YesNRC publication: Ye

Unusual Ether Lipids and Branched Chain Fatty Acids in Sea Cucumber (<i>Cucumaria frondosa</i>) Viscera and Their Seasonal Variation

The sea cucumber, Cucumaria frondosa, is harvested primarily for its muscular bands and body wall. Development of a nutraceutical product based on lipid recovered from its viscera would give commercial value to the entire organism; however, such development requires knowledge of the lipid and fatty acid (FA) profiles of the viscera. Here, we describe the lipid and FA composition of viscera recovered from C. frondosa harvested in coastal waters in the northwest Atlantic, taking into account variation due to harvest season. We found highest lipid content at ~29% in winter, with diacylglyceryl ethers (DAGE) comprising ~55% of the total lipid mass and triacylglycerols (TAG), phospholipids (PL) and monoacylglycerol ethers (MAGE) at 5–25% each. The branched chain FA, 12-methyltetradecanoic acid (12-MTA), represented 42% of total FA mass in DAGE. In summer, lipid content was lower at 24% and TAG was the dominate lipid, with proportions more than double that found in winter (45% vs. 20%); DAGE in summer dropped to ~30% of total lipids. In TAG, 12-MTA was much lower than found in DAGE in winter, at only 10% but eicosapentaenoic acid (EPA) content was ~20%, which brought the total EPA% to 28% of total FA—the highest among all three seasons. There was little effect of season on MAGE or PL proportions. These data can help harvesters maximize catch efforts in terms of lipid yield and profile

Comparison of antimicrobial activity in the epidermal mucus extracts of fish

The mucus layer on the surface of fish consists of several antimicrobial agents that provide a first line of defense against invading pathogens. To date, little is known about the antimicrobial properties of the mucus of Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp (Cyprinus carpio sub sp. koi), striped bass (Morone saxatilis), haddock (Melanogrammus aeglefinus) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were extracted with acidic, organic and aqueous solvents to identify potential antimicrobial agents including basic peptides, secondary metabolites, aqueous and acid soluble compounds. Initial screening of the mucus extracts against a susceptible strain of Salmonella enterica C610, showed a significant variation in antimicrobial activity among the fish species examined. The acidic mucus extracts of brook trout, haddock and hagfish exhibited bactericidal activity. The organic mucus extracts of brook trout, striped bass and koi carp showed bacteriostatic activity. There was no detectable activity in the aqueous mucus extracts. Further investigations of the activity of the acidic mucus extracts of brook trout, haddock and hagfish showed that these fish species had specific activity for fish and human pathogens, demonstrating the role of fish mucus in antimicrobial protection. In comparison to brook trout and haddock, the minimum bactericidal concentrations of hagfish acidic mucus extracts were found to be 3c 1.5 to 3.0 times lower against fish pathogens and 3c 1.6 to 6.6 folds lower for human pathogens. This preliminary information suggests that the mucus from these fish species may be a source of novel antimicrobial agents for fish and human health related applications.Peer reviewed: YesNRC publication: Ye

Selected bioactivities of vaccinium berries and other fruit crops in relation to their phenolic contents

Antioxidant activity, urinary tract protective activity, and cardioprotective anti-platelet effects are among the bioactivities associated with dietary phenolics. These bioactivities were measured in vitro in fruit extracts from seven Vaccinium species and five non-Vaccinium species to determine their relationship to total phenolic content and to anthocyanin and proanthocyanidin content. Berries belonging to the genus Vaccinium were particularly high in antioxidant activity and urinary tract protective anti-adhesion activity, while anti-platelet activity varied among species. There was a positive relationship between antioxidant activity (using the oxygen radical absorbing capacity (ORAC) assay) and both the total phenolic (R2 = 0.76) and anthocyanin content (R2 = 0.43) of the fruit, although there was no relationship between ORAC and proanthocyanidin content. There were no relationships between anti-adhesion activity and total phenolic content, anthocyanin content, or proanthocyanidin content. Likewise, no relationships were observed between anti-platelet activity and total phenolic content, anthocyanin content, or proanthocyanidin content. These results suggest that while antioxidant properties are characteristic of all fruit phenolics, in vitro anti-adhesion and anti-platelet bioactivities may be due to less abundant phenolic subgroups.Peer reviewed: YesNRC publication: Ye

Selected bioactivities of vaccinium berries and other fruit crops in relation to their phenolic contents

Antioxidant activity, urinary tract protective activity, and cardioprotective anti-platelet effects are among the bioactivities associated with dietary phenolics. These bioactivities were measured in vitro in fruit extracts from seven Vaccinium species and five non-Vaccinium species to determine their relationship to total phenolic content and to anthocyanin and proanthocyanidin content. Berries belonging to the genus Vaccinium were particularly high in antioxidant activity and urinary tract protective anti-adhesion activity, while anti-platelet activity varied among species. There was a positive relationship between antioxidant activity (using the oxygen radical absorbing capacity (ORAC) assay) and both the total phenolic (R2 = 0.76) and anthocyanin content (R2 = 0.43) of the fruit, although there was no relationship between ORAC and proanthocyanidin content. There were no relationships between anti-adhesion activity and total phenolic content, anthocyanin content, or proanthocyanidin content. Likewise, no relationships were observed between anti-platelet activity and total phenolic content, anthocyanin content, or proanthocyanidin content. These results suggest that while antioxidant properties are characteristic of all fruit phenolics, in vitro anti-adhesion and anti-platelet bioactivities may be due to less abundant phenolic subgroups.Peer reviewed: YesNRC publication: Ye

Improved methods of analysis for betaines in Ascophyllum nodosum and its commercial seaweed extracts

Beneficial effects of seaweeds and their extracts on crop performance have been attributed to a variety of compounds, including the betaines which are quaternary ammonium betaines. Methods of analysis of betaines published thus far suffer from low sensitivity, lack of baseline separation of individual betaines and from interference from other sample constituents. A rapid cleanup protocol and a sensitive LC- MS/MS method of analysis were developed to afford baseline separation of four betaines in the brown alga Ascophyllum nodosum and its commercial seaweed extract. Using this method, the presence of glycine betaine, \u3b4-aminovaleric acid betaine, \u3b3-aminobutyric acid betaine and laminine in A. nodosum, and commercial extracts derived from A. nodosum, were confirmed and quantified. The major betaine present was \u3b3-aminobutyric acid betaine accounting for 0.008-0.014% of the dry weight of the seaweed and 0.014-0.027% of the dry weight of the commercial extracts. Seasonal variation in betaine content was observed. Differences in the total betaine content were observed between A. nodosum of the yellow (0.011-0.017% dry weight) and the olive green (0.017-0.021% dry weight) coloured morphologies.Peer reviewed: YesNRC publication: Ye