Philosophiae Doctor - PhD / Recently, a great deal of interest has developed towards the isolation of bioactive compounds from marine sources due to their numerous health benefits. Furthermore, marine algae are valuable sources of structurally diverse metabolites with scientifically proven therapeutic claims. The cell walls are rich in sulfated polysaccharides such as fucoidans in brown algae, carrageenans in red algae and ulvans in green algae. These sulfated polysaccharides exhibit many beneficial biological activities such as anticoagulant, antiviral, antioxidative, anticancer and immunomodulating activities. They have great potential for further development as products in cosmeceutical, pharmaceutical and nutraceutical areas. Although the mechanism of action is still not clear, their biological activities could be mainly attributed to their major secondary metabolites namely; phlorotannins, terpenoids and fucoidans. There was use of comprehensive chromatographic separations and a full analysis of isolates using one or other of the spectroscopic techniques. Antioxidant and cytotoxicity tests were perfomed in details for Ecklonia maxima. Furthermore, structural and electronic features of the phlorotannins were compared in an attempt to provide an explanation for the differences in their radical scavenging properties. In this regard, two main radical scavenging mechanisms, hydrogen atom transfer (HAT) and electron transfer (ET), were assessed in order to determine the preferred mode of radical scavenging. Fully relaxed geometry optimizations of the neutral and the radical species were performed utilizing DFT/B3LYP and DFT/UB3LYP methods respectively. In further studies, the structural and functional properties of sulfated polysaccharides from the three brown and one red seaweeds were investigated. This was through detailed analysis of chemical composition of crude and purified polysaccharides using PMP - derivatization of hydrolysed sugars, anion exchange, molecular weight determination, ion chromatography , FT-IR, NMR to methylation analysis. The work reports isolation and characterization of compounds from four algae: Ecklonia maxima, phlorotannin derivatives, namely phloroglucinol (22), eckol (23), 7-phloroeckol (24), 2-phloroeckol (25) and a sterol, 24-ethylidine cholesterol (26); Splachinidium rugosum, 24-ethylidine cholesterol ( 26), 1, 3-Dicapryloyl-2-oleoylglycerol (27), E-3,7,11,15-tetramethylhexa dec-2-en-1-ol (phytol) (2 8); Macrocystis angustifolia, 24-ethylidine cholesterol (26); a red seaweed Aeodes orbitosa, and E -3, 7, 11, 15-tetramethylhexad ec-2-en-1-ol (28) and 17-(5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7, 8,9,11,12,14,15,16,17-dodecahydro-1 H-cyclopenta[a]phenanthren-3-ol (β-sitosterol) (29). Experimental findings and theoretical predictions of phlorotannins indicated that the radical scavenging activities followed the order 22< 23 < 25 < 24. Theoretical studies further indicated the ET mechanism is more significant than the HAT mechanism due to the high BDE values. Their polysaccharide structures were tentatively shown to have a backbone of (1-3) and (1-4) linkages with sulfate groups at O-2 and O-2, 3 positions. The only red algae studied contained, 2-O-methyl-D-galactose with (1-3) and (1-4)-glycosidic linkages possessing sulfate groups at positions 2 and 6.

Staphylococcus aureus is one of the major causes of food-borne pathogenesis. Antibiotic consumption for these pathogens has been increasing year by year world-wide. In order to decrease the use of synthetic antibiotics, fresh fruits and dry herbs consumed as beverages in regular diets were examined as potential natural antibiotics for the treatment of food based infections against Staphylococcus aureus. Herbs consumed as tea infusions including Pimpinella anisum L. (anise), Anthemis arvensis L. (camomile), Rosa canina L. (rosehip), Salvia fruticosa Mill (sage) and fresh fruit juices including Vitis vinifera L. (grape), Citrus sinensis L. (orange), Prunus persica L. (peach) and Punica granatum L. (pomegranate) were selected as samples of hot or cold consumed beverages in our daily diets. Extracts of fresh fruit juices were prepared in methanol, on the other hand, tea infusions of herbs were filtered and lyophilized. Antioxidant capacities of the plant samples were investigated by radical scavenging methods, namely 2&rsquo / 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) as well as determination of total phenolic and flavonoid contents. Furthermore, antimicrobial activities of plant samples were determined by minimum inhibitory concentration and minimum bactericidal concentration methods along with disc diffusion method. Trolox equivalent antioxidant capacities (TEAC) of the herbal tea infusions obtained by ABTS radical scavenging method were ranged between 48.38 &plusmn / 1.242 and 715.73 &plusmn / 4.265 (&mu / mol Trolox equivalent (TE)/g of extract) while, TEAC values of fresh fruits juices were between 26.86 &plusmn / 0.217 and 73.55 &plusmn / 0.973 (&mu / mol Trolox equivalent (TE)/g of extract). Moreover, EC50 values of the tea infusions obtained by DPPH radical scavenging method were ranged between 0.05 &plusmn / 0.001 and 1.53 &plusmn / 0.004 (mg/mL) while, EC50 values of the fruit juices were 1.6 &plusmn / 0.014 and 2 &plusmn / 0.093 (mg/mL). Total phenolic content of the plant samples tested in this study were varied from 1.383 (&mu / g gallic acid equivalent (GAE)/mg of extract) to 159.167 (&mu / g gallic acid equivalent (GAE)/mg of extract) and total flavonoid content varied from 0.111 (&mu / g quercetin equivalent (QE)/mg of extract) to 201.15 (&mu / g quercetin equivalent (QE)/mg of extract). Sage, orange and grape displayed higher antimicrobial activities with values of 1.5, 6 and 6 mg/mL minimum inhibitory concentrations and 1.5, 24 and 24 mg/mL minimum bactericidal concentrations, respectively. Inhibition zone diameters of sage, orange and grape were found to be 9, 9 and 11 mm.

Mangroves are the dominant vegetation in Everglades estuarine environment and are known to contain polyphenols such as tannins, which present similar fluorescence properties as some amino acid fluorophores. In the present study, gas chromatography–mass spectrometry (GC/MS) was used to quantify gallic acid, which is a normal monomer of polyphenols. The quantitative GC/MS analytical method was developed using gallic acid and tannic acid standards to quantify the false ‘protein-like’ fluorescence in DOM. The present study also compared the optical properties, reactive species (RS) production and radical scavenging ability of DOM from different regions of the Everglades and a correlation was observed between DOM composition and its photo-productivity. In general, the reactive species quantum yield decreased with increased DOM redox potential. The RS formation rates were controlled by the DOC and CDOM abundace. Normalized RS formation rates were shown to be influenced by DOM aromaticity and molecular weight characteristics

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The major objectives of this study were to investigate the antioxidant properties of flaxseed lignans secoisolariciresinol (SECO 2) and secoisolariciresinol diglycoside (SDG 1) and their major oxidative compounds using 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH 47) in an in vitro model of lipid peroxidation. This investigation was facilitated by the structural elucidation of the major oxidative compounds and the ability of flaxseed lignans to delay the onset of oxidation in two model systems. <p>This study showed that SECO 2 oxidation occurs at the aromatic (4-OH) and aliphatic (9-OH) hydroxyl groups. Conversely for SDG 1, only compounds derived from the oxidation of aromatic hydroxyl groups were obtained because the 9-OH position is glucosylated. <p>SECO 2 oxidation with AAPH 47 showed that the intermediate 2a is most likely involved in the generation of early-forming (48 and 52) and 2c for the formation of late-forming (49, 50 and 51) oxidation compounds. Compound 48 is formed from dimerization of 2a that is converted to 52 and then to 51. Compound 50 was formed by the addition of a carbon-centre free radical of AAPH (AP radical) to 2c. Compounds 50 and 51 trap carbon-centered AP radicals supporting SECO 2 as a chain-breaking antioxidant and AAPH 47 as a proper model for study of SECO 2 oxidation in vitro. <p>SDG 1 oxidation with AAPH 47 indicated that intermediates 1b and 1c are most likely involved for the formation of early forming compounds (55 and 58) and 1a leads to the late forming compounds (56 and 57). Compound 55 is a result of dimerization. Compound 56 may be directly formed via intermediate radical 1a by adding AP free radicals. Compound 56 was a stable non-radical compound that could trap AP free radicals, thereby supporting SDG 1 as a chain-breaking antioxidant. Hydrogen abstraction from 4-hydroxyl yielded the radical 1a and hydroxyl radical addition to 1a yielded 57. Compound 58 formed from the addition of OH or H2O to 1c. <p>This study demonstrated that AAPH 47 produces carbon-centred AP radicals upon thermal decomposition and mimics the formation of lipid peroxyl radicals. Interaction of carbon-centred AP radicals with SECO 2 and SDG 1 provides a good model to study the antioxidant reactions of SECO 2 in vitro. p*The relative antioxidant capacity of the flaxseed lignans versus BHT 17, in two model systems, was determined. The stoichiometric ratio for SECO 2 and SDG 1 were 1.5 and 1.1-1.2, respectively, compared to BHT 17 (2.0). The induction time by Rancimat analyzer measured inhibition of autoxidation mediated by flaxseed lignans SECO, SDG and SDG polymer in comparison with BHT 17. The induction time data demonstrated that SECO 2 protected canola oil better than either SDG 1 or SDG polymer 3. <p>These results are important for better understanding about the chemistry behind flaxseed lignan antioxidant activities. This study provided useful evidence that flaxseed lignans can be used as natural antioxidants.

The major objectives of this study were to investigate the antioxidant properties of flaxseed lignans secoisolariciresinol (SECO 2) and secoisolariciresinol diglycoside (SDG 1) and their major oxidative compounds using 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH 47) in an in vitro model of lipid peroxidation. This investigation was facilitated by the structural elucidation of the major oxidative compounds and the ability of flaxseed lignans to delay the onset of oxidation in two model systems. <p>This study showed that SECO 2 oxidation occurs at the aromatic (4-OH) and aliphatic (9-OH) hydroxyl groups. Conversely for SDG 1, only compounds derived from the oxidation of aromatic hydroxyl groups were obtained because the 9-OH position is glucosylated. <p>SECO 2 oxidation with AAPH 47 showed that the intermediate 2a is most likely involved in the generation of early-forming (48 and 52) and 2c for the formation of late-forming (49, 50 and 51) oxidation compounds. Compound 48 is formed from dimerization of 2a that is converted to 52 and then to 51. Compound 50 was formed by the addition of a carbon-centre free radical of AAPH (AP radical) to 2c. Compounds 50 and 51 trap carbon-centered AP radicals supporting SECO 2 as a chain-breaking antioxidant and AAPH 47 as a proper model for study of SECO 2 oxidation in vitro. <p>SDG 1 oxidation with AAPH 47 indicated that intermediates 1b and 1c are most likely involved for the formation of early forming compounds (55 and 58) and 1a leads to the late forming compounds (56 and 57). Compound 55 is a result of dimerization. Compound 56 may be directly formed via intermediate radical 1a by adding AP free radicals. Compound 56 was a stable non-radical compound that could trap AP free radicals, thereby supporting SDG 1 as a chain-breaking antioxidant. Hydrogen abstraction from 4-hydroxyl yielded the radical 1a and hydroxyl radical addition to 1a yielded 57. Compound 58 formed from the addition of OH or H2O to 1c. <p>This study demonstrated that AAPH 47 produces carbon-centred AP radicals upon thermal decomposition and mimics the formation of lipid peroxyl radicals. Interaction of carbon-centred AP radicals with SECO 2 and SDG 1 provides a good model to study the antioxidant reactions of SECO 2 in vitro. p*The relative antioxidant capacity of the flaxseed lignans versus BHT 17, in two model systems, was determined. The stoichiometric ratio for SECO 2 and SDG 1 were 1.5 and 1.1-1.2, respectively, compared to BHT 17 (2.0). The induction time by Rancimat analyzer measured inhibition of autoxidation mediated by flaxseed lignans SECO, SDG and SDG polymer in comparison with BHT 17. The induction time data demonstrated that SECO 2 protected canola oil better than either SDG 1 or SDG polymer 3. <p>These results are important for better understanding about the chemistry behind flaxseed lignan antioxidant activities. This study provided useful evidence that flaxseed lignans can be used as natural antioxidants.

<p>Traumatic brain injury (TBI) is a leading cause of death and disability TBI survivors often suffer from severe disturbances of cognition, memory and emotions. Improving the treatment is of great importance, but as of yet no specific neuroprotective treatment has been found. After TBI there are changes in ion homeostasis and protein regulation, causing generation of reactive oxygen species (ROS). Overproduction of ROS can lead to damage cellmembranes, proteins and DNA and secondary cell death. In the present thesis experimental TBI in rats were used to study the effects of the ROS scavengers α-phenyl-N-tert-butyl-nitrone (PBN) and 2-sulfophenyl-N-tert-butyl-nitrone (S-PBN) on morphology, function, intracellular signalling and apoptosis. </p><p>Posttreatment with PBN and S-PBN resulted in attenuation of tissue loss after TBI and S-PBN improved cognitive function evaluated in the Morris water maze (MWM). Pretreatment with PBN protected hippocampal morphology, which correlated to better MWM-performance after TBI.</p><p>To detect ROS-generation in vivo, a method using 4-hydroxybenzoic acid (4-HBA) microdialysis in the injured cortex was refined. 4-HBA reacts with ROS to form 3,4-DHBA, which can be quantified using HPLC, revealing that ROS-formation was increased for 90 minutes after TBI. It was possible to attenuate the formation significantly with PBN and S-PBN treatment. </p><p>The activation of extracellular signal-regulated kinase (ERK) is generally considered beneficial for cell survival. However, persistent ERK activation was found in the injured cortex after TBI, coinciding with apoptosis-like cell death 24 h after injury. Pretreatment with the MEK-inhibitor U0126 and S-PBN significantly decreased ERK activation and reduced apoptosis-like cell death. Posttreatment with U0126 or S-PBN showed robust protection of cortical tissue.</p><p>To conclude: ROS-mediated mechanisms play an important role in secondary cell death following TBI. The observed effects of ROS in intracellular signalling may be important for defining new targets for neuroprotective intervention.</p>

Traumatic brain injury (TBI) is a leading cause of death and disability TBI survivors often suffer from severe disturbances of cognition, memory and emotions. Improving the treatment is of great importance, but as of yet no specific neuroprotective treatment has been found. After TBI there are changes in ion homeostasis and protein regulation, causing generation of reactive oxygen species (ROS). Overproduction of ROS can lead to damage cellmembranes, proteins and DNA and secondary cell death. In the present thesis experimental TBI in rats were used to study the effects of the ROS scavengers α-phenyl-N-tert-butyl-nitrone (PBN) and 2-sulfophenyl-N-tert-butyl-nitrone (S-PBN) on morphology, function, intracellular signalling and apoptosis. Posttreatment with PBN and S-PBN resulted in attenuation of tissue loss after TBI and S-PBN improved cognitive function evaluated in the Morris water maze (MWM). Pretreatment with PBN protected hippocampal morphology, which correlated to better MWM-performance after TBI. To detect ROS-generation in vivo, a method using 4-hydroxybenzoic acid (4-HBA) microdialysis in the injured cortex was refined. 4-HBA reacts with ROS to form 3,4-DHBA, which can be quantified using HPLC, revealing that ROS-formation was increased for 90 minutes after TBI. It was possible to attenuate the formation significantly with PBN and S-PBN treatment. The activation of extracellular signal-regulated kinase (ERK) is generally considered beneficial for cell survival. However, persistent ERK activation was found in the injured cortex after TBI, coinciding with apoptosis-like cell death 24 h after injury. Pretreatment with the MEK-inhibitor U0126 and S-PBN significantly decreased ERK activation and reduced apoptosis-like cell death. Posttreatment with U0126 or S-PBN showed robust protection of cortical tissue. To conclude: ROS-mediated mechanisms play an important role in secondary cell death following TBI. The observed effects of ROS in intracellular signalling may be important for defining new targets for neuroprotective intervention.