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Bioprospecting for bioactive polysaccharides from marine algae endemic to South AfricaJanuary, Grant Garren January 2016 (has links)
>Magister Scientiae - MSc / Fucoidan is a marine-derived sulphated polysaccharide with bioactive properties ideal for the food, chemical and pharmaceutical industries. The polysaccharide consists largely of L-fucose, has a highly heterogeneous structure and is of diverse origin. Fucoidan was extracted from Ecklonia maxima, Laminaria pallida and Splachnidium rugosum and the effect of different extraction methods on fucoidan heterogeneity was assessed. Extraction methods employed hot water, hydrochloric acid or calcium chloride salt. Fucoidan yield and purity were determined by various colorimetric assays. Highest fucoidan yield was obtained with the hot water extraction method as seen by highest L-fucose content. Splachnidium rugosum extracts contained ~5 times more L-fucose than Ecklonia maxima and Laminaria pallida extracts. The salt extraction method yielded extracts free of contaminants, however L-fucose content in all extracts was >20 times lower. Acid extraction yielded highest levels of uronic acid contamination and liberated sulphate from the fucoidan polysaccharide. The fucose-to-sulphate ratio for Ecklonia maxima was approximately 1:5, whilst the ratios for Splachnidium rugosum and Laminaria pallida were approximately 1:1 and 1:2, respectively. The acid and salt extraction methods removed all traces of protein contaminants, while the hot water method retained very low levels of protein. The extraction method used to isolate fucoidan was a determining factor in yield and purity. Chemical compositional analyses of hot water extracts were assessed by gas chromatography mass spectroscopy. Splachnidium rugosum and Laminaria pallida extracts consisted largely of L-fucose, while Ecklonia maxima fucoidan was characterized with high glucose abundance. Crude hot water and acid extracts from Splachnidium rugosum tissue were fractionated and purified by (anionic) ion exchange chromatography as bioactivity has been correlated to lower molecular weight forms. In water extracts, ion exchange chromatography resulted in close to 90% decrease in L-fucose, sulphate and uronic acid, while protein content increased by 57%. Similar results were reported for acid extracts; however protein content did not change significantly. These results show that method of extraction may affect the composition of fucoidan post-purification. Hot water extraction is recommended due to higher fucoidan yield, as reflected by L-fucose content, and higher sulphate-to-fucose ratio. High protein content after ion exchange chromatography was however of concern. Since mucilage in Splachnidium rugosum thallus was free of protein, fucoidan was precipitated from mucilage with ethanol. Fucoidan yield of mucilage was >15-fold higher than content in purified hot water extracts with a sulphate-to-fucose ratio of ~1:1. The average molecular weight of native fucoidan in mucilage was estimated at 2367 kDa. The polysaccharide was hydrolysed by gamma-irradiation levels of 10-50 kGy to fractions ranging between 60 and 15.5 kDa. Hot water crude fucoidan extracts from Ecklonia maxima, Laminaria pallida, and Splachnidium rugosum were assessed for anti-oxidant activity by measuring the ability to scavenge free radicals and the capacity to reduce copper ions with 2,2-Diphenyl-1-picrylhydrazyl and Cupric Reducing Anti-oxidant Capacity assays, respectively. Ecklonia maxima crude fucoidan displayed highest anti-oxidant activity and capacity, having the potential to scavenge reactive oxygen species as well as the capacity to reduce copper to less toxic forms in mammalian systems. Splachnidium rugosum showed weakest anti-oxidant activity and lowest reducing capacity. The anti-cancer activity of crude and purified hot water Splachnidium rugosum extracts, as well as non-irradiated (native) and gamma-irradiated fucoidan, and commercially procured fucoidan were assessed for anti-cancer activity against MCF-7 breast cancer cells. Splachnidium rugosum crude and purified fucoidan displayed a half maximal inhibitory concentration of 0.7 mg/mL and 0.029 mg/mL, respectively. Low cytotoxicity of crude and purified Splachnidium rugosum fucoidan against non-cancerous breast epithelial cell line MCF-12A was observed, as seen by half maximal inhibitory concentration values of 2 mg/mL and 0.663 mg/mL, respectively. The cancer specific selectivity of purified Splachnidium rugosum fucoidan was therefore much higher as reflected by 10-fold higher selectivity index than that of crude fucoidan. Native and low molecular weight gamma-irradiated fucoidan also showed bioactive properties including anti-cancer activity as seen by the reduction of cell proliferation in vitro, whereas crude fucoidan showed the ability to scavenge free radicals, and the capacity to reduce copper ions. / National Research Foundation (NRF)
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Structural investigation of the natural products composition of selected South African seaweeds.Maina, Mwangi Henry January 2014 (has links)
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.
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Proteome signature of breast cancer cells treated with fucoidanJanodien, Fatima January 2016 (has links)
>Magister Scientiae - MSc / Breast cancer is responsible for a large portion of cancer-related deaths. Worldwide, incidence is increasing. Routinely-used treatments for breast cancer are invasive and are associated with a range of side-effects which may affect quality of life. Fucoidan, a marine bioactive compound, found primarily in brown seaweed, has various medicinal qualities. Among its bioactivities studied, it has potent anticancer activity. Despite numerous studies, the mechanism of action of fucoidan on cancer cells remains unclear. This project aims to shed light on the mechanism of action of fucoidan by studying its effect on the MCF7 breast cancer cell proteome. The IC50 obtained for fucoidan treated MCF7 cells was 0.2 mg/ml. Decrease in expression of XIAP and phosphorylation of ERK1/2 was observed, indicating a decrease in inhibition of apoptosis and increased sensitivity to apoptosis, respectively. Literature reports activation of several caspases, including caspase-3, in various cell lines after to fucoidan treatment. Taken together, with data from the current study it can be said that fucoidan treatment led to cell death by apoptosis. SILAC analysis identified over 2000 proteins with more than 1700 at 95% confidence. STRING analysis of enriched proteins revealed 19 cell death related proteins. However, SILAC results were ambiguous with regards to differential protein regulation and should be repeated with lower electrospray ionization flow rates, pairwise and single sample runs, and validation with Western blot analysis of various apoptosis related proteins and biochemical assays. / National Research Foundation
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Intérêt de la vectorisation du t-PA au thrombus : exemple du Fucoidan aminé / Interest of vectorization of t-PA to the thrombus : example of the amino FucoidanGhebouli, Radouane 20 December 2016 (has links)
Les accidents vasculaires cérébraux (AVC) ischémiques, l’infarctus du myocarde (IDM) et l’embolie pulmonaire (EP) sont la première cause de mortalité et de morbidité dans le monde. Leur prise en charge est limitée à la recanalisation des vaisseaux occlus par le thrombus. La thrombolyse intraveineuse par le rt-PA (l’activateur tissulaire du plasminogène recombinant) est le seul traitement médicamenteux utilisé dans ces situations d’urgences vasculaires. En dépit de ces bénéfices, cette thérapie s’entrave de plusieurs effets délétères principalement d’un risque d’hémorragie cérébrale. Augmenter le rendement fibrinolytique du rt-PA via sa vectorisation sans augmenter ses effets délétères serait d’un grand intérêt. Le Fucoidane, un polysaccharide anionique extrait principalement des algues brunes, ayant une affinité de l’ordre du nanomolaire à la P-sélectine, a démontré un ciblage spécifique du thrombus in vivo. Dans notre travail, nous avons développé une molécule bipolaire associant le Fucoidane comme vecteur au thrombus, aminé par des lysines en N–terminales pour lier le rt-PA exogène. Le potentiel de vectorisation du t-PA par le Fucoidane aminé ainsi que la preuve du concept ont été validé par notre travail. Nous avons montré sur des modèles de thrombi in vitro une accélération de lyse des thrombi, et in vivo une augmentation de l’efficacité thrombolytique du t-PA, couplé au Fucoidan aminé, par injection périphérique. / Acute ischemic stroke (AIS), acute myocardial infarction (AMI), and pulmonary embolism (PE) are the main cause of mortality and morbidity worldwide. Thrombolysis by intravenous injection (IV) of recombinant tissue plasminogen activator (rt-PA) remains the most common non-interventional treatment to recanalize vessels occluded. However, Its use is limited by significant drawbacks, including bleeding complications. Recent studies showed that Fucoidan (sulfated polysaccharides extracted from brown algae) target in vivo intraluminal thrombus.In this study, we have developed a bipolar construct associating Fucoidan, able to target the thrombus, and a NH2 exposing lysine platform able to bind to t-PA. We hypothesize that this construct was able to vectorize t-PA to the thrombus, would increase its fibrinolytic efficacy and avoid its deleterious effects. In vitro assays, t-PA complexed to amino Fucoidan has shown its over thrombolytic superiority as compared to tPA alone. In vivo mesenteric, arterial and deep vena cava thrombosis and thrombolysis, vascular reperfusion was significantly increased in mice treated with amio Fucoidan complexed to t-PA as compared in mice treated by t-PA alone. This data demonstate that amino Fucoidan is an excellent vector of t-PA to the thrombus.
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Atividades anticoagulante e antioxidante de extratos brutos ricos em polissacar?deos sulfatados das macroalgas marinhas marrons Canistrocarpus cervicornis, Dictyota mertensii e Dictyopteris delicatula e de Heterofucanas de Canistrocarpus cervicornisC?mara, Rafael Barros Gomes da 28 October 2010 (has links)
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Previous issue date: 2010-10-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In the present study, extracts rich-sulfated polysaccharides were obtained from three different species of Dictyotales (a class of brown macroalgae): Canistrocarpus cervicornis, Dictyota mertensii and Dictyopteris delicatula and their anticoagulant and antioxidant activities were evaluated. All extracts showed anticoagulant activity on aPTT assay, but not on PT assay. Extracts also exhibited total antioxidant activity, superoxide radical scavenging capacity and ferric chelating property. The extract from C. cervicornis showed the best results and was choose to have their sulfated polysaccharides fractioned and subsequently analysed. Thus, six fractions (CC-0.3, CC-0.5, CC-0.7, CC-1.0, CC-1.2 and CC-2.0) were obtained by proteolysis followed by sequential acetone precipitation. Agarose gel eletrophoresis stained with blue toluidine, confirmed the presence of sulfated polysaccharides in all fractions. Chemical analyses showed that all fractions presented heterofucans mainly constitued by fucose, galactose, glucuronic acid and sulfate. Any fraction changed the PT. However, all fractions were able to double the aPTT on a dose-dependent manner. CC- 0.3, CC-0.5, CC-0.7 and CC-1.0 needed only 0.100 mg/mL to double the aPTT, result only 1.25 times higher than the Clexane? (0.080 mg/mL), a commercial low molecular heparin. The heterofucans presented appreciable total antioxidant capacity, low capacity on scavenging hydroxyl radical and good efficiency on scavenging superoxide radicals (except CC-1.0). CC-1.2 showed 43.1 % on superoxide radical scavenging. This result was higher than that showed by the same concentration of gallic acid (41.8 %), a known antioxidant. Furthermore, the heterofucans showed excelent activity on ferrous chelating activity (except CC-0.3). CC-0.5, CC-0.7 and CC-1.0 showed the highest activities with 47.0 % of ferrous chelating activity, a result 2.0 times lesser than that exhibited by the same concentration of EDTA. These results clearly indicated the beneficial effects of heterofucans extracted from C. cervicornis as potential anticoagulant and antioxidant agents. However additional steps of purification, structural studies, besides in vivo experiments are needed for these fucans may be used as therapeutic agents / No presente estudo, extratos brutos ricos em polissacar?deos sulfatados foram obtidos a partir de tr?s esp?cies de Dictyotales (uma ordem de macroalgas marrons): Canistrocarpus cervicornis, Dictyota mertensii e Dictyopteris delicatula, e suas atividades anticoagulante e antioxidante foram avaliadas. Todos os extratos apresentaram atividade anticoagulante frente ao ensaio de aPTT, mas n?o sobre o ensaio de PT. Os extratos tamb?m exibiram atividade antioxidante total, capacidade em sequestrar radicais super?xido e propriedade de quelar ferro. O extrato obtido a partir de C. cervicornis apresentou os melhores resultados e foi escolhido para ter seus polissacar?deos sulfatados fracionados e subsequentemente analisados. Deste modo, seis fra??es (CC-0.3, CC-0.5, CC-0.7, CC-1.0, CC-1.2 e CC-2.0) foram obtidas por prote?lise seguida de precipita??o sequencial com acetona. Eletroforese em gel de agarose corada com azul de toluidina comprovou a presen?a de polissacar?deos sulfatados em todas as fra??es. As an?lises qu?micas mostraram que todas as fra??es apresentam heterofucanas constitu?das principalmente por fucose, galactose, ?cido glucur?nico e sulfato. Nenhuma fra??o alterou o PT. Entretanto todas as fra??es foram capazes de dobrar o aPTT de uma maneira dose dependente. As fra??es, CC-0.3, CC-0.5, CC-0.7 e CC-1.0, precisaram de apenas 0,100 mg/mL para dobrar o tempo de coagula??o, concentra??o que ? apenas 1,25 vezes maior do que aquela utilizada com Clexane? (heparina de baixo peso molecular) para se obter o mesmo efeito. As heterofucanas apresentaram apreci?vel capacidade antioxidante total, baixa capacidade em sequestrar radicais hidroxila e uma boa efici?ncia em sequestrar radicais super?xido (exceto CC-1.0). CC-1.2 mostrou uma capacidade de sequestrar 43,1 % dos radicais super?xido. Este resultado foi maior do que o apresentado pela mesma concentra??o de ?cido g?lico (41,8 %), um antioxidante conhecido. Al?m disso, as heterofucanas mostraram uma excelente atividade em quelar ferro (exceto CC-0.3). CC-0.5, CC-0.7 e CC-1.0 apresentaram as maiores atividades com 47,0 % de quela??o f?rrica, um resultado 2 vezes menor do que o exibido pela mesma concentra??o de EDTA. Estes resultados indicaram claramente os efeitos ben?ficos de heterofucanas extra?das de C. cervicornis como potenciais agentes anticoagulante e antioxidante. Entretanto, passos adicionais de purifica??o, estudos estruturais, al?m de experimentos in vivo, s?o necess?rios para que estas fucanas possam vir a ser utilizadas como agentes terap?uticos
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Efeito do Fucoidam de Fucus vesiculosus em um modelo experimental de artrite reumat?ideXavier, Caroline Addison Carvalho 11 October 2005 (has links)
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Previous issue date: 2005-10-11 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Rheumatoid arthritis (RA) is systemic auto imune disorder. It is caracterized by chronic inflammation of joints leading to progressive erosion of cartilage and bone. We investigated the effect of the administration of fucoidan, sulfated polysaccharides, from algae Fucus vesiculosus in the acute (6h) in zymosan-induced arthritis (AZy). Wistar rats (180-230 g) were used for all groups experimental. Non-treated animals received just intraarticular injection of 1 mg the zymosan, control group received intraarticular injection of 50 ?L the saline, groups received either fucoidan of Fucus vesiculosus (15, 30, 50 or 70 mg/Kg) or parecoxib (1 mg/Kg) 1 hour after injection of zymosan. After 6 h, the articular exudates were collected for evaluation of the cell influx and nitrite (Griess reaction) release. The sinovial membranes and articular cartilages were excised for histopathological analysis and by determination of the glycosaminoglycan (GAG), respectively. ZyA led to increased NO and cell influx into the joints. Therapeutic administration of the fucoidan or parecoxib did significantly inhibited the cell influx and the synovitis, as compared to non-treated rats (p<0,05), though being able to reduced NO release. Representative agarose gel electrophoresis of the GAGs, the content of condroitin-sulphate was observed during the process. These findings suggest that the fucoidan from Fucus vesiculosus has potential anti-inflammatory activity / A artrite reumat?ide (AR) ? uma doen?a auto-imune sist?mica, caracterizada por inflama??o cr?nica das articula??es, resultando em progressiva eros?o cartilaginosa e ?ssea. Neste trabalho foi investigado o efeito da administra??o do fucoidam, polissacar?deos sulfatados, da alga marinha Fucus vesiculosus na fase aguda (6h) da artrite induzida por zymosan (AZy). Ratos Wistar (180-230 g) foram utilizados para todos os grupos experimentais. Animais n?o tratados receberam apenas 1 mg de zymosan intraarticular (i.a), o grupo controle recebeu 50 ?L de solu??o salina intraarticular (i.a.), os outros grupos receberam fucoidam de Fucus vesiculosus (15, 30, 50 ou 70 mg/Kg,) ou parecoxib (1 mg/Kg), por via intraperitoneal (i.p.) 1 hora ap?s a inje??o de zymosan (i.a.). Ap?s 6 h, o exsudato articular foi coletado para an?lises do influxo celular e libera??o de nitrito (reagente de Griess). As membranas sinoviais e as cartilagens articulares foram retiradas para an?lises histopatol?gicas e para a determina??o dos glicosaminoglicanos, respectivamente. A AZy caracterizou-se por libera??o aumentada de NO e influxo de c?lulas inflamat?rias, nas juntas. A administra??o terap?utica do fucoidam ou parecoxib inibiu (p<0,05) o influxo celular, a libera??o de ?xido n?trico e a sinovite, comparado ao grupo n?o tratado. Por eletroforese em gel de agarose e tamp?o PDA 0,05M pH9,0 foi observado bandas com migra??es semelhantes ao condroitim sulfato (CS). Estes resultados sugerem que o fucoidam de Fucus vesiculosus tem um potencial anti-inflamat?rio
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Atividade anticoagulante, antioxidante e antitumoral de heterofucanas da alga Dictyopteris delicatula (Lamouroux, 1809)Magalhaes, Kaline Dantas 02 December 2011 (has links)
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Previous issue date: 2011-12-02 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / In the present study, six families of sulfated polysaccharides were obtained from seaweed Dictyopteris delicatula (Lamouroux, 1809) and their anticoagulant, antioxidant and antitumor activities were evaluated. All fractions showed anticoagulant activity on aPTT assay, but not on PT assay. Fractions also exhibited total antioxidant activity, superoxide radical scavenging capacity and ferric chelating property. Thus, six fractions (F0.5v, F0.7v, F1.0v, F1.3v, F1.5v e F2.0v) we obtained by proteolytic digestion, followed by acetone fractionation and molecular sieving on Sephadex G-100. Chemical analyses demonstrated that all polysaccharides contain heterofucans composed mainly of fucose, xylose, glucose, galactose, uronic acid, and sulfate. Any fractions changed the PT. However, all fractions were able on double the aPPT on a dose-dependent manner. The heterofucans F0.7v and F1.0v showed low anticoagulant activity while F1.5v presented the most prominent anticoagulant activity .When compared to Clexane?, a low molecular weight heparin, at same concentration F1.5v presented similar anticoagulant activity. The fucans F0.5v and F0.7v at 1.0 mg/mL showed high ferric chelating activity (~45%), whereas fucans F1.3v (0.5 mg/mL) showed considerable reducing power, about 53.2% of the activity of vitamin C. The fucan F1.5v presented the most prominent anticoagulant activity. The best antiproliferative activity was found with fucans F1.3v and F0.7v. However, F1.3v activity was much higher than F0.7v inhibiting almost 100% of HeLa cell proliferation. These fucans have been selected for further studies on structural characterization as well as in vivo experiments, which are already in progress / No presente estudo, seis popula??es de polissacar?deos sulfatados foram obtidas a partir da alga Dictyopteris delicatula (Lamouroux, 1809) e suas atividades anticoagulante, antioxidante e antitumoral avaliadas. Deste modo, as seis fra??es (F0.5v, F0.7v, F1.0v, F1.3v, F1.5v e F2.0v) foram obtidas por prote?lise seguida por fracionamento de acetona e gel filtra??o molecular Sephadex G-100. Todas as fra??es apresentaram atividade anticoagulante frente ao ensaio de aPTT, mas n?o sobre o ensaio de PT. As heterofucanas exibiram atividade antioxidante total, capacidade em sequestrar radicais super?xido e propriedade de quelar ferro. As an?lises qu?micas demonstraram que todos os polissacar?deos cont?m heterofucanas composta principalmente por fucose, xilose, glicose, galactose, ?cido ur?nico, e sulfato. Nenhuma das fra??es alterou o PT. Entretanto, todas as fra??es foram capazes de dobrar o aPTT de uma maneira dose dependente. As heterofucanas F0.7v e F1.0v demonstraram baixa atividade anticoagulante, enquanto a F1.5v apresentou a maior atividade anticoagulante e quando comparada com Clexane?, uma heparina de baixo peso molecular, em mesma concentra??o, apresentou antividade anticoagulante semelhante. As fucanas F0.5v e F0.7v a 1,0 mg/mL mostraram alta atividade de quela??o ferica (~ 45%), enquanto a fucana F1.3v (0,5 mg/mL) mostrou consider?vel potencial redutor, cerca de 53,2% da atividade da vitamina C. A melhor atividade antitumoral foi encontrada nas fucanas F1.3v e F0.7v. No entanto, a atividade F1.3v foi muito superior a F0,7v, quase 100% de inibi??o da prolifera??o de c?lulas HeLa (c?lula de c?ncer de colo de ?tero). Estas fucanas foram selecionadas para novos estudos sobre caracteriza??o estrutural, bem como em experimentos in vivo, que j? est?o em andamento / 2020-01-01
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Fucana ativa via das Map quinases e inibe a prolifera??o de c?lulas de ov?rio de hamster chin?s (CHO)Nobre, Leonardo Thiago Duarte Barreto 08 July 2011 (has links)
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Previous issue date: 2011-07-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Fucan is a term used to denominate L-fucose rich sulfated polysaccharides. The fucans have been studied due their pharmacological activities like antithrombotic, antiproliferative and antioxidant. We have extracted three fucan fractions from the brown seaweed Spatoglossum schr?ederi. These fucans were denominated Fuc B 1, Fuc B 1.5 and Fuc B 2. The chemical analyzes show that the fucans have very similar composition as demonstrated by agarose electrophoresis gel, sugar and sulfate content. The antiproliferative effect was determined by MTT and BrdU methodologies in CHO cells. The inhibition of proliferation effect of the three fractions was about 40%. Therefore this we proceed just with the Fuc B 2 due the higher yield. There is no apoptosis indication using the anexin V/propidium iodide test. We found a cell cycle phase G1 arrest. The western blotting show that the PKC; pFAK; pERK 1/2 are activated when the cells were treated with fucans. The treatement with inhibitor of MAPK PD98059 extinguished the fucan effect. These results indicates that fucan act by the ERK pathway inducing the cell death. / Fucana ? um termo utilizado para denominar polissacar?deos sulfatados ricos em L-Fucose. As fucanas t?m sido estudadas devido suas atividades farmacol?gicas: antitromb?tica, antiproliferativa e antioxidante. N?s extra?mos tr?s fra??es de fucanas da alga Spatoglossum schr?ederi. Essas fucanas, denominadas de Fuc B 1, Fuc B 1.5 e Fuc B 2, apresentam uma composi??o muito similar como demonstrado pela eletroforese em gel de agarose, e conte?do de a??car e sulfato. O efeito antiproliferativo foi determinado pelas metodologias de MTT e BrdU em c?lulas CHO. O efeito na inibi??o da prolifera??o das tr?s fra??es foi de cerca de 40%. Assim, procedemos somente com a Fuc B 2 devido seu maior rendimento. N?o houve indica??o de apoptose usando a marca??o com anexina V-FITC/ Iodeto de Prop?deo. Identificamos uma parada na fase G1 do ciclo celular. Os ensaios de western blotting mostraram que PKC, pFAK e pERK 1/2 s?o ativadas quando as c?lulas s?o tratadas com Fuc B. O tratamento com o inibidor de MAPK PD98059 aboliu o efeito da fucana. Esses efeitos indicam que a fucana age via ERK para inibir a prolifera??o das c?lulas.
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Stratégies de thérapie pro-angiogénique de l’artériopathie oblitérante des membres inférieurs / Pro-angiogenic therapy strategies of peripheral arterial diseaseSapharikas, Elène 01 October 2015 (has links)
L’artériopathie oblitérante des membres inférieurs conduit progressivement au rétrécissement des artères qui assurent la vascularisation des membres inférieurs. Il en résulte une ischémie des tissus irrigués par ces artères et à terme, en cas d’occlusion artérielle, une ischémie critique conduisant à une amputation du membre. De nouvelles stratégies de thérapie cellulaire basées sur l’injection de cellules progénitrices capables d’induire une angiogenèse thérapeutique se sont développées ces dernières années. Cependant le faible taux d’incorporation des cellules transplantées dans le tissu ischémique limite le développement de ces nouvelles approches. Dans ce contexte, mon travail de thèse a consisté à étudier deux approches thérapeutiques distinctes pouvant améliorer les thérapies pro-angiogènes. La première étude porte sur le fucoïdane, polysaccharide sulfaté d’origine naturelle, antithrombotique favorisant la formation de nouveaux vaisseaux sanguins dans le modèle murin d’ischémie du membre inférieur. Nous avons montré qu’il induisait le recrutement de monocytes en améliorant leur adhésion à l’endothélium activé en condition dynamique, ainsi que leur adhésion à la matrice et leur transmigration in vitro. Cette action est médiée par l’activation des voies de signalisation ERK et p38 et la sécrétion de métalloprotéinases 9. De plus, le fucoïdane entraine une polarisation des macrophages de type pro-angiogènes in vitro. Il augmente leur recrutement dans le muscle ischémié permettant de réduire ainsi la phase inflammatoire post-ischémique, la nécrose et de favoriser le processus de cicatrisation. La deuxième étude porte sur le rôle des neuropilines (NRP), co-récepteurs du VEGF (facteur de croissance pro-angiogène) exprimés à la surface des ECFC, afin de comprendre leur implication au niveau moléculaire dans le mécanisme d’action pro-angiogène des ECFCs et optimiser l’efficacité de la thérapie cellulaire. A l’aide du système d’extinction par ARN interférent, nous avons découvert un mécanisme de compensation jamais étudié auparavant puisque l’inhibition de NRP1 entraine une augmentation de celle de NRP2 et une diminution de la prolifération et de la migration des ECFCs. En revanche, l’extinction de NRP2 n’a pas d’effet sur l’expression de NRP1, mais induit une augmentation de l’adhésion des ECFCs à la matrice extracellulaire associée à une augmentation de la phosphorylation des ERK1/2. / Vascular diseases such as Peripheral Arterial Disease may evolve towards critical limb ischemia, requiring revascularization or amputation. New strategies of cell therapy based on the injection of progenitor cells able to induce therapeutic angiogenesis have been recently developed. However the low level of incorporation of transplanted cells in the ischemic tissue limits the development of these new approaches. In this context, my thesis was to study two different therapeutic approaches that can improve the pro-angiogenic therapies. The first focuses on fucoidan, a marine sulphated polysaccharide with antithrombotic properties. We have previously shown promising angiogenic properties of fucoidan in vivo. We found that fucoidan increases monocyte recruitment and improves their adhesion to activated endothelium under dynamic condition. It also increases in vitro transmigration. This action is mediated by the activation of ERK and p38 signaling pathways and metalloproteinase 9 secretion. Further, fucoidan can lead macrophage polarization to the pro-angiogenic type in vitro. It increases macrophage recruitment in ischemic muscle that could reduce post-ischemic inflammatory phase and necrosis leading to healing process. The second study focuses on the role of neuropilin (NRP), co-receptors of VEGF (pro-angiogenic growth factor). The aim of this part was to understand their involvement in the pro-angiogenic properties of ECFC at molecular level and optimize the efficiency of cell therapy. Using siRNA, we found a compensation mechanism never studied before. The NRP1 inhibition leads to an increase in the NRP2 expression and a decrease of ECFC proliferation and migration. The NRP2 silencing has no impact on NRP1, but induces ECFC adhesion to the extracellular matrix correlated with an increased level of ERK1 / 2 phosphorylation.
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Stratégies de thérapie pro-angiogénique de l’artériopathie oblitérante des membres inférieurs / Pro-angiogenic therapy strategies of peripheral arterial diseaseSapharikas, Elène 01 October 2015 (has links)
L’artériopathie oblitérante des membres inférieurs conduit progressivement au rétrécissement des artères qui assurent la vascularisation des membres inférieurs. Il en résulte une ischémie des tissus irrigués par ces artères et à terme, en cas d’occlusion artérielle, une ischémie critique conduisant à une amputation du membre. De nouvelles stratégies de thérapie cellulaire basées sur l’injection de cellules progénitrices capables d’induire une angiogenèse thérapeutique se sont développées ces dernières années. Cependant le faible taux d’incorporation des cellules transplantées dans le tissu ischémique limite le développement de ces nouvelles approches. Dans ce contexte, mon travail de thèse a consisté à étudier deux approches thérapeutiques distinctes pouvant améliorer les thérapies pro-angiogènes. La première étude porte sur le fucoïdane, polysaccharide sulfaté d’origine naturelle, antithrombotique favorisant la formation de nouveaux vaisseaux sanguins dans le modèle murin d’ischémie du membre inférieur. Nous avons montré qu’il induisait le recrutement de monocytes en améliorant leur adhésion à l’endothélium activé en condition dynamique, ainsi que leur adhésion à la matrice et leur transmigration in vitro. Cette action est médiée par l’activation des voies de signalisation ERK et p38 et la sécrétion de métalloprotéinases 9. De plus, le fucoïdane entraine une polarisation des macrophages de type pro-angiogènes in vitro. Il augmente leur recrutement dans le muscle ischémié permettant de réduire ainsi la phase inflammatoire post-ischémique, la nécrose et de favoriser le processus de cicatrisation. La deuxième étude porte sur le rôle des neuropilines (NRP), co-récepteurs du VEGF (facteur de croissance pro-angiogène) exprimés à la surface des ECFC, afin de comprendre leur implication au niveau moléculaire dans le mécanisme d’action pro-angiogène des ECFCs et optimiser l’efficacité de la thérapie cellulaire. A l’aide du système d’extinction par ARN interférent, nous avons découvert un mécanisme de compensation jamais étudié auparavant puisque l’inhibition de NRP1 entraine une augmentation de celle de NRP2 et une diminution de la prolifération et de la migration des ECFCs. En revanche, l’extinction de NRP2 n’a pas d’effet sur l’expression de NRP1, mais induit une augmentation de l’adhésion des ECFCs à la matrice extracellulaire associée à une augmentation de la phosphorylation des ERK1/2. / Vascular diseases such as Peripheral Arterial Disease may evolve towards critical limb ischemia, requiring revascularization or amputation. New strategies of cell therapy based on the injection of progenitor cells able to induce therapeutic angiogenesis have been recently developed. However the low level of incorporation of transplanted cells in the ischemic tissue limits the development of these new approaches. In this context, my thesis was to study two different therapeutic approaches that can improve the pro-angiogenic therapies. The first focuses on fucoidan, a marine sulphated polysaccharide with antithrombotic properties. We have previously shown promising angiogenic properties of fucoidan in vivo. We found that fucoidan increases monocyte recruitment and improves their adhesion to activated endothelium under dynamic condition. It also increases in vitro transmigration. This action is mediated by the activation of ERK and p38 signaling pathways and metalloproteinase 9 secretion. Further, fucoidan can lead macrophage polarization to the pro-angiogenic type in vitro. It increases macrophage recruitment in ischemic muscle that could reduce post-ischemic inflammatory phase and necrosis leading to healing process. The second study focuses on the role of neuropilin (NRP), co-receptors of VEGF (pro-angiogenic growth factor). The aim of this part was to understand their involvement in the pro-angiogenic properties of ECFC at molecular level and optimize the efficiency of cell therapy. Using siRNA, we found a compensation mechanism never studied before. The NRP1 inhibition leads to an increase in the NRP2 expression and a decrease of ECFC proliferation and migration. The NRP2 silencing has no impact on NRP1, but induces ECFC adhesion to the extracellular matrix correlated with an increased level of ERK1 / 2 phosphorylation.
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