Ministério Da Educação Universidade Federal Do Rio Grande Do Norte Centro De Ciências Da Saúde Programa De Pós-Graduação Em Ciências Da Saúde

MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

ATIVIDADE ANTICOAGULANTE IN VITRO DE GALACTANAS SULFATADAS OBTIDAS DA ALGA VERDE UDOTEA FLABELLUM (J.ELLIS & SOLANDER) M.HOWE

MAXSUELL LUCAS MENDES MARQUES

NATAL/RN 2018

MAXSUELL LUCAS MENDES MARQUES

ATIVIDADE ANTICOAGULANTE IN VITRO DE GALACTANAS SULFATADAS OBTIDAS DA ALGA VERDE UDOTEA FLABELLUM (J.ELLIS & SOLANDER) M.HOWE

Dissertação apresentada ao Programa de Pós-Graduação em Ciência da Saúde da Universidade

Federal do Rio Grande do Norte como requisito para a obtenção do título de Mestre em Ciências da Saúde.

Orientador: Hugo Alexandre de Oliveira Rocha

NATAL/RN 2018

Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial do Centro Ciências da Saúde - CCS

Marques, Maxsuell Lucas Mendes. Atividade anticoagulante in vitro de galactanas sulfatadas obtidas da alga verde Udota flabellum (J.Ellis & Solander) M.Howe / Maxsuell Lucas Mendes Marques. - Natal, 2018. 37f.: il.

Dissertação (Mestrado) - Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Programa de Pós-graduação em Ciências da Saúde. Natal, RN, 2018. Orientador: Hugo Alexandre de Oliveira Rocha.

1. Alga Marinha (Alga verde) - Dissertação. 2. Polissacarídeos Sulfatados - Dissertação. 3. Anti-IIa - Dissertação. 4. Anticoagulante - Dissertação. I. Rocha, Hugo Alexandre de Oliveira. II. Título.

RN/UF/BS-CCS CDU 582.263

Elaborado por ANA CRISTINA DA SILVA LOPES - CRB-15/263

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MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

Coordenadora do Programa de Pós-graduação em Ciências da Saúde - Prof. Dra. Ana Katherine da Silveira Gonçalves de Oliveira.

MAXSUELL LUCAS MENDES MARQUES

ATIVIDADE ANTICOAGULANTE IN VITRO DE GALACTANAS SULFATADAS OBTIDAS DA ALGA VERDE UDOTEA FLABELLUM (J.ELLIS & SOLANDER) M.HOWE

Aprovada em ___ / ___ / ______

Banca examinadora:

Presidente da banca: Hugo Alexandre de Oliveira Rocha

Membros da banca:

AGRADECIMENTOS

A Deus, por aparar em suas mãos nos dias mais difíceis, por me dar força e determinação e por me manter firme diante dos desafios. Obrigado por me permitir errar e aprender sempre durante essa “infinita” jornada. Ao “Chefe”, Prof. Dr. Hugo Alexandre, que com seus métodos poucos ortodoxos, conseguiu ser mais que um grande mentor, mas um grande amigo e conselheiro, me ajudando a formar além de um pesquisador melhor a cada dia, uma pessoa mais integra, responsável, criativa e colaborativa. Meus eternos agradecimentos. As grandes orientadoras Dayanne, Jailma, Mariana, Marília, Monique e Ruth, pela paciência, pelos eternos experimentos. Tenham certeza que cada ensinamento, brincadeiras e conselhos foram e estão guardados comigo. Nunca esquecerei todo o apoio e carinho de vocês. Aos meus grandes amigos, que mesmo sem cerveja e sem café nunca me abandonaram. Almino, Arthur, Fernando, Gabriel, Leo, Pablo, Popó, Rafael, Raniere, Rony e Vinicius, todos vocês são responsáveis por grandes alegrias e ensinamentos, nesta labuta incessante que é desenvolver o conhecimento científico. A minha família acima de tudo, meu irmão Samuel, por ser minha inspiração de todos os dias, me dando força e alegria para jamais explodir diante de tanta pressão. A minha rainha, minha Mãe, Iris, por nunca ter desistido de mim, dos meus sonhos, por me apoiar, lutar comigo todos os dias por um amanhã melhor, sem nunca descansar. Sem vocês eu nada seria, meu amor eterno a vocês. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq pelo suporte financeiro (Edital Universal, n° 408369/2016-7); A Coordenação de Aperfeiçoamento Pessoal de Nível Superior-CAPES por suportar financeiramente este trabalho por meio do Programa Ciências do Mar (AUXPE-CIMAR-1956/2014) e do Programa Nacional de Cooperação Acadêmica (CAPES/PROCAD n° 2965/2014).

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Não te deixes destruir… Ajuntando novas pedras e construindo novos poemas. Recria tua vida, sempre, sempre. Remove pedras e planta roseiras e faz doces. Recomeça. Faz de tua vida mesquinha um poema. E viverás no coração dos jovens e na memória das gerações que hão de vir. Esta fonte é para uso de todos os sedentos. Toma a tua parte. Vem a estas páginas e não entraves seu uso aos que têm sede.

(Cora Coralina)

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RESUMO

O principal fármaco anticoagulante conhecido é o polissacarídeo sulfatado conhecido como heparina. No entanto, o uso contínuo da heparina pode causar efeitos colaterais, como o desenvolvimento de trombocitopenia, embolia arterial e complicações hemorrágicas. Portanto, há uma busca por novos anticoagulantes que tenham menos efeitos colaterais do que a heparina. Muitas algas sintetizam polissacarídeos sulfatados com atividade anticoagulante. Por isso, neste trabalho, extratos ricos em polissacarídeos sulfatados, e que ainda não foram avaliados como anticoagulantes, foram obtidos de 22 algas tropicais (4 vermelhas, 11 marrons e 7 verdes) encontradas no nordeste do Brasil e avaliadas como agentes anticoagulantes. Quinze extratos apresentaram atividade anticoagulante, incluindo todos os extratos de algas verdes. O extrato da alga verde Udotea flabellum foi o mais potente, necessitando apenas de 3 µg para dobrar o tempo de coagulação plasmática no teste do tempo de tromboplastina parcial ativada (TTPA). Resultado semelhante foi obtido com 1 µg de heparina. Duas homogalactanas sulfatadas denominadas de F-I (130 kDa) e F-II (75 kDa) com alta atividade anticoagulante foram obtidos a partir deste extrato com o uso de diferentes etapas de purificação bioguiadas. Suas atividades anticoagulantes estavam próximas às da heparina. F-I e F-II (0,5-10 µg/mL) não foram capazes de inibir diretamente a trombina. Porém, na presença de antitrombina, a F-I (0,5 μg/mL) foi mais eficaz que a heparina (0,5 μg/mL) na inibição da trombina, enquanto a F-II apresentou efeitos similares à heparina. Em conjunto, os resultados fornecem fortes evidências para o potencial anticoagulante dos homogalactanas sulfatados de U. flabellum.

Palavras-chave: Algas marinhas tropicais; polissacarídeos sulfatados; anti-IIa.

ABSTRACT

The main known anticoagulant drug is the sulfated polysaccharide named heparin. However, the use of heparin can cause side effects such as the development of thrombocytopenia, arterial embolism, bleeding complications. Therefore, new anticoagulants that have fewer side effects than heparin are being sought. Many seaweeds are known to synthesize sulfated polysaccharides with anticoagulant activity. Therefore, in this paper, sulfated polysaccharide-rich extracts were obtained from 22 tropical seaweeds (4 red, 11 brown, and 7 green) found in northeastern Brazil and evaluated as anticoagulant agents. Fifteen extracts had anticoagulant activity, including all extracts of green seaweeds. Udotea flabellum extract was the most potent, requiring only 3 µg to double the plasma coagulation time in the activated partial thromboplastin time (APTT) test. A similar result was obtained with 1 µg of heparin. Two sulfated homogalactans F-I (130 kDa) and F-II (75 kDa) with high anticoagulant activity were obtained from this extract using several bio-guided purification steps. Their anticoagulant activities were close to that of heparin. F-I and F-II (0.5–10 μg/mL) were not able to directly inhibit thrombin. In the presence of antithrombin, F-I (0.5 μg/mL) was more effective than heparin (0.5 μg/mL) in inhibiting thrombin, while F-II showed similar effects as heparin. Taken together, the results provide strong evidence for the anticoagulant potential of sulfated homogalactans from U. flabellum.

Keywords: Tropical marine seaweeds; sulfated polysaccharides; anti-IIa.

LISTA DE ABREVIATURAS E SIGLAS

Anti IIa ativado...... Trombina ativada aPTT...... Tempo de tromboplastina parcial ativada AT...... Antitrombina cm...... Centímetro °C...... Grau centrígrado Fator IIa...... Fibrina ativada pela trombina FCS...... Soro fetal bovino F-I and FI-II…...... Homogalactanas purificadas da alga Udotea flabellum FT...... Fator tecidual g/mg/µg...... Grama/Miligrama/Micrograma x g...... vezes a gravidade h...... Hora HCII...... Cofator II da heparina HCl...... Ácido clorídrico HS...... Heparan sulfato IIa...... Trombina kDa...... Kilodalton L/mL/µL...... Litro/Mililitro/Microlitro M/nM...... Molar/Nanomolar mg...... Miligrama NaCl...... Cloreto de sódio NaOH...... Hidróxido de sódio nd...... Não detectado nm...... Nanômetro PBS...... Solução salina tamponada com fosfato pH...... Potencial hidrogênio iônico PS...... Polissacarídeo sulfatado PT...... Tempo de protrombina RN...... Rio Grande do Norte S ...... Sul TFPI...... Inibidor da via do fator tecidual TT...... Tempo de Trombina UF...... Udotea flabellum UF-0.3/UF-0.5/UF -0.6/UF -0.7/UF-1.0/UF-2.0……. Frações obtidas da alga Udotea flabellum USA...... Estados Unidos da América vs...... versus W...... Oeste Xa...... Fator X ativado

LISTA DE FIGURAS

Figura 01 Representação esquemática dos complexos procoagulantes...... 13 Figura 02 Mecanismos de ação anticoagulante dos polissacarídeos sulfatados de algas marinhas...... 16 Figura 03 Foto da alga verde Udotea flabellum...... 17

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SUMÁRIO

1. INTRODUÇÃO...... 13 2. OBJETIVOS...... 18 2.1. OBJETIVO GERAL...... 18 2.2. OBJETIVOS ESPECÍFICOS ...... 18 3. JUSTIFICATIVA ...... 19 4. MÉTODOS ...... 20 5. ARTIGOS PRODUZIDOS ...... 21 5.1. ARTIGO ...... 22 6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES...... 38 7. REFERÊNCIAS...... 39

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1 INTRODUÇÃO

Coagulação e anticoagulantes O processo de coagulação envolve elementos próximos às lesões como as paredes endoteliais, plaquetas e proteínas solúveis. No tocante as estas últimas, foi proposto em meados dos anos 60 por MacFarlane, Davie e Ratnoff [1,2] um modelo denominado de cascata da coagulação, no qual o mecanismo de coagulação é dividido inicialmente em duas vias: via intrínseca, assim chamada porque todos os componentes estão presentes no sangue; e via extrínseca, cujo fator tecidual (FT), uma proteína da membrana plasmática de células endoteliais, se faz necessário, além de componentes circulantes, para esta via ocorra. O ponto de convergência das vias extrínseca e intrínseca é conhecido como via comum da coagulação e é caracterizada pela ativação de fibrinogênio à fibrina ativada pela trombina (Fator IIa), formando uma malha de fibrina que vai ser estabilizada pelo fator XIIIa [3]. No entanto, tal modelo é inadequado já que esta divisão em vias intrínseca e extrínseca não ocorre in vivo. Com base nisso, um novo modelo constituído por 3 complexos procoagulantes: complexo tenase extrínseco, complexo tenase intrínseco e complexo protrombinase foi proposto e é atualmente aceito pela comunidade científica (Figura 01) [4].

Figura 01. Representação esquemática dos complexos procoagulantes. (a) O Fator VII encontra-se ativado em concentrações muito baixas no sangue. (b) O Fator VIIa tem grande afinidade pelo FT exposto em membranas celulares, inclusive de plaquetas. O complexo FT/VIIa formado é capaz de ativar uma quantidade consideravelmente maior de Fator VII. (c) O principal papel de FT/VIIa, no entanto, é formar um complexo com os Fatores X e IX (Complexo tenase extrínseco), que promove a ativação desses dois fatores. Uma vez ativado, o Fator Xa, leva a formação de pequenas concentrações de trombina, que por sua vez ativam os cofatores V e VIII. (d) Tanto o fator VIIIa quanto fator IXa se complexam ao Fator X (Complexo tenase intrínseco) ativando-o, o que por conseguinte leva a ativação de mais moléculas de trombina. (e) Os Fatores Va e Xa se complexam ao Fator II, formando o Complexo protrombinase, que aumenta significativamente sua conversão em trombina (a

formação de trombina por este complexo é 50 vezes maior do que pelos demais). Por fim, a ativação da trombina irá gerar a conversão de fibrinogênio em fibrina. FT: Fator tecidual. Fonte: Câmara [5].

Em condições fisiológicas, a coagulação sanguínea é estritamente regulada por anticoagulantes endógenos para que não haja uma ativação indesejada, o que levaria a uma coagulação intravascular disseminada, resultando na formação excessiva de trombos. As principais proteínas regulatórias que funcionam como anticoagulantes naturais são: Os inibidores fisiológicos da coagulação (anticoagulantes naturais) de maior relevância fisiológica são a antitrombina (AT), também chamada de antitrombina III pela literatura mais antiga; TFPI (inibidor da via do fator tecidual); proteína C e proteína S [6]. Em várias situações patológicas os anticoagulantes naturais são superados por agentes ativadores da cascata de coagulação o que passa a exigir a utilização de anticoagulantes exógenos, o mais conhecido é a heparina. Um polissacarídeo sulfatado, da classe dos glicosaminoglicanos, extraído de mastócitos encontrados em intestinos suínos ou pulmões bovinos [7,8]. A heparina possui diversos mecanismos de ação que favorecem sua ação anticoagulante. O principal mecanismo de ação da heparina reside em sua capacidade de se complexar com a AT e aumentar sua a afinidade com a trombina. Além disso, o complexo Heparina/Antitrombina é capaz de interagir com os fatores IXa, Xa, XIa e XIIa, inativando-os. Por fim, a heparina ainda é capaz de formar um complexo ternário com o cofator II da heparina (HCII) e a trombina, o que resulta na inibição dessa última mediada por HCII [9]. A heparina é o único polissacarídeo sulfatado utilizado comercialmente como anticoagulante, entretanto sua utilização pode provocar reações adversas tais como: trombocitopenia (mediada por anticorpos), osteopenia (devido à ligação da heparina a osteoblastos que liberam fatores ativadores de osteoclastos) e efeito hemorrágico residual [10]. Além disso, entre 2007 e 2008, uma série de mortes e efeitos adversos severos foram associados a pacientes que receberam injeções de heparina nos EUA e países europeus. Esse evento que ficou conhecido como “A crise da heparina” ocorreu devido à contaminação de formulações chinesas do fármaco contaminadas por condroitin supersulfatado artificialmente [11]. Por último, a síntese da heparina sintética, mesmo de oligossacarídeos de heparina, ainda é uma possibilidade muito distante [12].

Dentro deste contexto, a busca por novos compostos anticoagulantes que possam ser uma alternativa ao uso da heparina vem se intensificado e um dos principais alvos são os polissacarídeos sulfatados. Tanto os sulfatados quimicamente [13,14] como os sulfatados naturalmente, como outros glicosaminoglicanos de animais [15], e polissacarídeos sulfatados (PS) de algas marinhas [16,17]. Os PS de algas marinhas têm uma grande variedade de atividades biológicas, mas a sua ação anticoagulante é a mais amplamente estudada [18]. Apesar do modelo de coagulação sanguínea em “cascata” ter sido considerado inadequada do ponto vista fisiológico, a divisão do processo de coagulação em vias ainda é utilizada para interpretação de testes laboratoriais que avaliam a coagulação sanguínea. Por conseguinte, a atividade anticoagulante dos PSs é geralmente avaliada por esses testes, como: tempo de protrombina (PT) e tempo de tromboplastina parcial ativada (aPTT), que avaliam se o polissacarídeo age nas vias extrínseca e intrínseca da coagulação, respectivamente; e pelo tempo de trombina (TT), que é o teste para a via comum da coagulação, que avalia a formação de fibrina mediada por trombina [19]. A ação anticoagulante dos polissacarídeos sulfatados reside, principalmente, na potencialização dos inibidores naturais da trombina e fator Xa: a AT e o HCII. Esses PS podem agir por dois mecanismos distintos: induzindo a mudança alostérica nas serpinas (AT e HCII) ou a cadeia do PS pode atuar como uma ''ponte'', aproximando a protease à serpina [20]. No entanto, há casos de PS que exibem um efeito anticoagulante independente de serpinas, por exemplo, inibindo diretamente a atividade da trombina [21,22]. O mecanismo de ação dos PS sob as proteases trombina e fator Xa pode ser avaliado por testes in vitro utilizando substratos cromogênicos na presença ou ausência de serpinas [19]. Na Figura 2 sumariza-se os mecanismos de ação anticoagulante dos PS de algas marinhas.

Figura 02 - Mecanismos de ação anticoagulante dos polissacarídeos sulfatados de algas marinhas. PS – polissacarídeo sulfatado, X – fator X inativo, Xa – fator X ativo, Va – fator V ativo, AT – antitrombina, HCII – cofator II da heparina, TFPI – inibidor da via do fator tecidual, HS – heparan sulfato.

Fonte: Adaptado de Costa [23].

A alga Udotea flabellum

A alga verde Udotea flabellum (J.Ellis & Solander) M.Howe 1904 (Figura 3) tem como sinonimia os nomes Corallina flabellum J.Ellis & Solander 1786, Udotea flabellata J.V.Lamououx 1816 e Udotea halimeda Kützing 1849. Ela é encontrada em várias partes do globo, como Austrália, Estados Unidos, Cuba e outras ilhas caribenhas e Argentina. No Brasil, ela ocorre ao longo de todo o seu litoral [24]. Apesar dessa ampla distribuição há poucos artigos cientificos sobre essa alga. No tocante a seu PSs, há apenas um artigo sobre esse tema [25]. Foi demonstrado que a U. flabellum sintetiza galactanas sulfatadas. Porém esses polímeros apresentam uma peculariedade em relação a outras galactanas sulfatadas, o fato de apresentarem grupos sulfatos ligados ao carbono 2 de alguns resíduos de galactose. Além disso, elas apresentam resíduos de galactose que também são piruvatados (substituição entre o carbanos 4 e 6) ou dissulfatados, no caso, também nos carbonos 4 e 6 [25].

Figura 03: Alga verde Udotea flabellum (J.Ellis & Solander). Fonte: http://bermudaconservation.squarespace.com/aquatic -plants-algae/

2 OBJETIVOS

2.1 Objetivo geral

Objetivou-se nesse trabalho purificar de forma bioguiada um ou mais polissacarídeo(s) sulfatado(s) anticoagulante(s) sintetizado(s) por algas marinhas encontradas no litoral do Rio Grande do Norte.

2.2 Objetivo específicos

1. Obter extratos ricos em polissacarídeos sulfatados de 22 algas marinhas encontradas no litoral do Rio Grande do Norte e avalia-lo com agentes anticoagulantes; 2. Obter frações ricas em polissacarídeos sulfatados da alga cujo o extrato apresentou maior atividade anticoagulante e avalia-las como agentes anticoagulantes; 3. A partir da fração com maior atividade anticoagulante purificar um ou mais polissacarídeos sulfatados com alta atividade anticoagulante e caracteriza-las no tocante ao seu teor de polissacarídeos, sulfato e contaminantes proteicos, bem como, com relação a sua composição monossacarídica e massa molecular aparente; 4. Avaliar o efeito dos PSs purificados em teste in vitro de inibição da trombina, na presença ou não de AT e HCII.

3 JUSTIFICATIVA

O grupo de pesquisa em se insere esta dissertação demonstrou que extratos ricos em PS de sete algas do litoral do Rio Grande do Norte apresentavam atividade anticoagulante, pois afetavam o tempo de coagulação no teste de APTT [26]. Posteriormente, demonstrou-se que extratos ricos em PS das algas Dictyopetris justii [27] e Gracilaria birdiae [28], também algas do litoral do Rio Grande do Norte, apresentavam atividade anticoagulante quando avaliados no teste de APTT. Contudo, há mais de 80 algas do litoral do RN que ainda não tiveram seus PS avaliados como agentes anticoagulantes. Deve-se também ter em mente cada alga sintetiza pelo menos um tipo de polissacarídeo e que esse pode não ser encontrado em mais nenhum outro tipo de ser vivo deste planeta. O que faz desse PS um composto único cujas as propriedades podem diferir dos demais PS já estudados [29]. Portanto este trabalho foi realizado com intuito de ampliar o conhecimento sobre o potencial anticoagulante de PS de algas marinha encontradas no litoral do Rio Grande do Norte, bem como, trazer a luz da ciência, o conhecimento sobre características de um ou mais PS com atividade anticoagulante purificado de uma dessas algas.

4 MÉTODOS

Os métodos e materiais utilizados para o desenvolvimento dessa dissertação estão descritos no capítulo 5.

5 ARTIGOS PRODUZIDOS

5.1 O artigo “Anticoagulant activity of sulfated galactans from the tropical green seaweed Udotea flabellum” foi submetido no periódico Molecules, que possui fator de impacto 3.009 (2017) e Qualis B1/2017 da CAPES, para área de Medicina II.

Article Anticoagulant activity of sulfated galactans from the tropical green seaweed Udotea flabellum

Maxsuell Lucas Mendes Marques1,2,, Fernando Bastos Presa1,2,, Rony Lucas Silva Viana1, Mariana Santana Santos Pereira Costa3, Daniel Lima Bellan4, Monique Gabriela Chagas Faustino Alves1, Leandro Silva Costa3, Edvaldo Silva Trindade4 and Hugo Alexandre Oliveira Rocha1,2,*

1 Laboratório de Biotecnologia de Polímeros Naturais- BIOPOL, Departamento de Bioquímica, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte-RN 59078-970, Brazil; [email protected] (M.L.M.M.); [email protected] (R.L.S.V.); [email protected] (F.B.P.); [email protected] (M.G.C.F.A) 2 Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte 59078-970, Brazil; 3 Instituto Federal de Educação, Ciência, e Tecnologia do Rio Grande do Norte (IFRN), Rio Grande do Norte 59500-000, Brazil; [email protected] (M.S.S.P.C), [email protected] (L.S.C.) 4 Departamento de Biologia Celular, Universidade Federal do Paraná, Curitiba, Paraná-PR, 80060-000, Brazil; [email protected] (E.S.T); [email protected] (D.L.B.)

* Correspondence: [email protected]; Tel.: +55-84-3215-3416

Received: date; Accepted: date; Published: date

Abstract: The main known anticoagulant drug is the sulfated polysaccharide named heparin. However, the use of heparin can cause side effects such as the development of thrombocytopenia, arterial embolism, bleeding complications. Therefore, new anticoagulants that have fewer side effects than heparin are being sought. Many seaweeds are known to synthesize sulfated polysaccharides with anticoagulant activity. Therefore, in this paper, sulfated polysaccharide-rich extracts were obtained from 22 tropical seaweeds (4 red, 11 brown, and 7 green) found in northeastern Brazil and evaluated as anticoagulant agents. Fifteen extracts had anticoagulant activity, including all extracts of green seaweeds. Udotea flabellum extract was the most potent, requiring only 3 µg to double the plasma coagulation time in the activated partial thromboplastin time (APTT) test. A similar result was obtained with 1 µg of heparin. Two sulfated homogalactans F-I (130 kDa) and F-II (75 kDa) with high anticoagulant activity were obtained from this extract using several bio-guided purification steps. Their anticoagulant activities were close to that of heparin. F-I and F-II (0.5–10 μg/mL) were not able to directly inhibit thrombin. In the presence of antithrombin, F-I (0.5 μg/mL) was more effective than heparin (0.5 μg/mL) in inhibiting thrombin, while F-II showed similar effects as heparin. Taken together, the results provide strong evidence for the anticoagulant potential of sulfated homogalactans from U. flabellum.

Keywords: Tropical marine seaweeds; sulfated polysaccharides; anti-IIa.

1. Introduction In the 1960s, it became clear how multiple factors interact to convert prothrombin to thrombin, resulting in the formation of a fibrin clot. In 1964, two independent groups presented evidence on how these multiple factors interact and proposed two very similar models that resemble the idea of a coagulation cascade. In both cases, coagulation occurred through a series of steps in which activation of each of the coagulation factors (factors X; IX; VIII, VII) leads to the activation of another, culminating in the formation of thrombin (factor II) [1]. In this model, coagulation is divided into three stages: the intrinsic, extrinsic and common pathways that are evaluated by the activated partial thromboplastin time (APTT) tests, prothrombin

23 time (PT) tests, and thrombin time (TT) tests, respectively. Although the coagulation cascade model is no longer accepted as it does not reflect how coagulation occurs physiologically [2], the use of its concept persists because these tests are cheap and easy to perform and therefore can be used to study the early stages of coagulopathies and to discover new anticoagulant compounds. The main known anticoagulant drug is the sulfated polysaccharide (SP) named heparin [3]. However, the use of heparin can cause side effects such as the development of thrombocytopenia, arterial embolism, bleeding complications, and even death of the patient [4]. In addition, the problem of heparin deliberate contamination with supersulfated chondroitin can induce death of the patient [5]. Therefore, new anticoagulants that have fewer side effects than heparin are being sought. SPs from seaweeds have been studied more in this context. Many sulfated seaweed polysaccharides have anticoagulant activity [6] that is not exclusively dependent on the sulfate content of SP. SP activity depends on its negative charge, specifically, on the distribution of this charge throughout the molecule; this influences how SP interacts with clotting factors and natural inhibitors, such as antithrombin (AT) and heparin cofactor II (HCII), which determines whether or not blood anticoagulation is promoted [7]. A feature of seaweed SP is that each type of seaweed synthesizes at least one SP that is unique and therefore may be one that is more suitable than that currently used for a certain application. With this in mind, in this paper, the SPs from 21 tropical seaweeds found in northeastern Brazil were extracted and evaluated as anticoagulant agents.

2. Results and discussion

2.1. Evaluation of the anticoagulant activity of SP-rich extract from tropical seaweed The SP-rich extracts were obtained from the tropical seaweeds described in the Methods section and their anticoagulant activities were determined by the APTT method. The results are shown in Table 1; more than 50% of the extracts succeeded in altering the plasma coagulation time. However, the activity (in terms of the amount (µg) required to double APTT compared to saline control) was not the same for all the extracts; for example, the extract of G. birdiae was effective at the high amount of 100 µg. Six extracts, all obtained from brown seaweed, showed activity when they were present above 10 µg. All extracts obtained from green seaweed had good anticoagulant activity; in particular, U. flabellum extract required only 3 µg to double plasma coagulation time.

Table 1. Anticoagulant activity of SP-rich extracts obtained from several tropical seaweed.

APTT

Green seaweeds µg* C. cupressoides 7.0 ± 0.8 C. sertulariodes 6.0 ± 0.8 C. prolifera 8.0 ± 0.7 C. racemosa 8.0 ± 0.5 C. isthmocladum 10.0 ± 0.7 U. flabellum 3.0 ± 0.3 U. lactuca 10.0 ± 0.9 Brown seaweeds L. variegata 25.0 ± 1.5 S. vulgare 20.0 ± 1.9 P. gymnospora 50.0 ± 2.5 S. felipendula nd S. schröederi nd

D. mertensii 35.0 ± 4.7 D. delicatula 45.0 ± 1.8 D. menstrualis Nd C. cervicornis 15.0 ± 0.9 D. justii nd D. ciliolata nd Red seaweeds G. Birdiae 100.0 ± 5.0 G. caudata nd A. multifida nd A. especifera nd Heparin 1.0

*Data are reported as amount (µg) required to double APTT compared to saline control. APTT = activated partial thromboplastin time; nd =anticoagulant activity not detected until 200 µg.

The SPs of the green seaweed U. flabellum had the highest anticoagulant activity. This was not surprising given that Arata et al. [8] had already shown that SPs from this seaweed has anticoagulant activity. However, the anticoagulant activity of Udotea SP presented in their study was much lower than that observed in our study. The structures of seaweed SPs can vary depending on environmental factors, as shown by Siddhanta et al. [9]. These authors showed that the yield, monosaccharide composition, and amount of SP from the green seaweed Ulva fasciata varied seasonally. It is possible that the difference between the anticoagulant activity reported by Arata et al. [8] and us can be explained by seasonal variations. As U. flabellum SP showed the most potent activity in APTT anticoagulant tests, we chose this SP for subsequent steps.

2.2. Obtaining the SP-rich fractions from the seaweed U. flabellum In order to identify the anticoagulant SP within the Udotea extract, we fractionated the extract by adding increasing volumes of acetone as described above. After adding two volumes of acetone, no further precipitates formed. Thus, six SP-rich fractions from the green seaweed U. flabellum were obtained when 0.3, 0.5, 0.6, 0.7, 1.0, and 2.0 volumes of acetone were used. These fractions were named according to the acetone volume used to obtain them, i.e., UF-0.3, UF-0.5, UF -0.6, UF -0.7, UF-1.0, and UF-2.0, respectively. The fractionation yield and the anticoagulant activity of these fractions are shown in Table 2. The fraction UF-0.5v yielded the highest quantity (~ 34%), followed by fraction UF-2.0v (~20%). On the other hand, the lowest yield was obtained with the fraction UF-0.3v (~ 5%). Recently, using this same fractionation protocol, Presa et al. [10] also obtained six SP-rich fractions from U. flabellum with very similar yields to those observed in Table 2. U. flabellum belongs to the Bryopsidales order, and two other seaweeds of this order, Codium isthmocladum [11] and Caulerpa cupressoides [12], also underwent acetone fractionation, resulting in 5 and 4 SP-rich fractions from these seaweeds, respectively. These results indicate that this class of seaweed synthesizes more than one type of SP.

Table 2. Yield and anticoagulant activity of SP-rich fractions extracted from the U. flabellum

Yielda APTTb Udotea Fractions (%) (µ) UF-0.3 5.8 20.0 UF-0.5 33.8 2.5 UF-0.6 10.3 2.5 UF-0.7 14.1 5.0

UF-1.0 14.8 20.0 UF-2.0 21.2 nd aAll polysaccharides obtained by acetone precipitation were dried and weighed and total mass of each sample corresponded to 100%. bData are reported as amount (µg) required to double APTT compared to saline control. APTT = activated partial thromboplastin time; nd =anticoagulant activity not detected until 200 µg.

2.3. Purification of SPs from the UF-0.5 fraction

Fraction UF-0.5 yielded the most material precipitated with acetone; also, it was the fraction that presented with the highest anticoagulant activity. Therefore, this fraction was chosen to purify SP with anticoagulant activity. SPs of UF-0.5 were purified according to their molecular mass as described above; thus, five SPs were obtained that were named F-I, F-II, F-III, F-IV, and F-V. The results of the chemical analysis and the APTT of these SPs are summarized in Table 3.

Table 3. Chemical composition and anticoagulant activity of polysaccharides extracted from U. flabellum Molar Ratio Sugar Sulfate Protein APTTb SPs Yield (%)a (%) (%) (%) (µ) Gal Glu Man Xyl URSF 75.2 ± 2.1 18.2 ± 0.5 5.0 ± 1.8 3.0 1.0 1.2 0.5 0.3 UF-0.5 82.0 ± 0.9 18.7 ± 0.8 0.6 ± 0.3 2.5 1.0 0.5 - - F-I 62.2 ± 2.6 84.0 ± 1.6 14.0 ± 1.1 nd 1.0 1.0 - nd nd F-II 25.1 ± 1.3 76.0 ± 2.8 24.0 ± 0.5 nd 1.0 1.0 - nd nd F-III 7.0 ± 0.6 72.2 ± 1.8 2.0 ± 0.3 8.0 ± 0.7 5.0 1.0 1.0 0.1 - F-IV 3.1 ± 0.3 58.2 ± 3.5 3.0 ± 0.8 14 ± 1.3 1.0 1.0 1.0 0.1 0.2 F-V 3.0 ± 0.2 66.5 ± 1.8 3.0 ± 0.4 17 ± 0.9 5.0 1.0 0.8 1.0 - a All samples obtained were dried and weighed, and total mass corresponded to 100%. bData are reported as amount (µg) required to double aPTT compared to saline control. Heparin (1.0 µg) was required to double APTT compared to saline control. Gal: galactose; Xyl: xylose; Man: mannose; Gluc: glucose; -Traces; nd—not detected. USRF - Udotea SP-rich fraction

The presence of xylose among the monosaccharides composing the samples is due to the 3-linked β-D-xylans replacing cellulose in U. flabellum [13]. Mannose and glucose have previously been described as components of SP-rich extracts obtained from Udotea seaweed [8]. However, the amounts previously reported of these monosaccharides are much lower than what we report. This difference can be explained by the seasonal differences, as well as by the extraction method, since we used a different extraction method from that used in the previous paper. Several authors have reported that different methods of extraction of SP yield different monosaccharide compositions [14]. Galactose was the predominant monosaccharide in all samples, with F-I and F-II being composed almost exclusively of this monosaccharide. This agrees with data in the literature. Although there is not much data on the structure of SPs from order Bryopsidales, the main SP described in these seaweeds are sulfated galactans [11-12; 15-16]. All of these SPs showed high anticoagulant activity, especially F-I, F-II and F-IV. However, F-III, F-IV, and F-V showed low amounts of sulfate and polysaccharide, although they were highly contaminated by proteins in comparison to F-I and F-II. For these two SPs, similar to heparin, only 1 µg was required to double the coagulation time. Notably, anticoagulant activity does not depend on the sulfate content of SP, since F-II, with twice the sulfate content of F-I, presented similar anticoagulant activity as F-I. This corroborates other reports finding that the anticoagulant activity seems to depend not only on the amount of sulfate found in SP, but also on other characteristics, such as the arrangement of these functional groups within the structure of the polysaccharide [14;17]. The APTT data show a great deal of potential for F-I and FII. However, it was necessary to know the degree of purity of these compounds. Therefore, in order to verify whether F-I and F-II consist of

26 single compounds, they were subjected to gel filtration chromatography. As shown in Figure 1, F-I and FI-II produced only one peak in each chromatogram, indicating that these SPs consist of only one polysaccharide. In addition, it was possible to determine the apparent molecular weight of the samples, 130 kDa and 75 kDa for F-I and FII, respectively.

Figure 1. Gel permeation chromatography of SPs from U. flabellum. F-I and F-II were applied to a Sephadex G-100 column (140 x 1 cm). The column was eluted with 0.2 M acetic acid, 1 mL fractions were collected, and the fractions were analyzed for the presence of sugars by the Dubois method and metachromasia, as describe in the Methods section.

2.4. Inhibitory Effect of F-I and F-II on Thrombin Activity (Anti-IIa activity) Table 3 shows the high anticoagulant activity of F-I and F-II. In general, the literature reports that the anticoagulant mechanism of action of SP from seaweed is based on the inhibition of thrombin by different mechanisms. Thus, to identify the anticoagulant mechanism of F-I and F-II, their inhibitory effects on thrombin were evaluated. In the direct thrombin inhibition assay, the polysaccharides were tested at concentrations not exceeding 10 μg/mL, because above this concentration, a precipitate was formed. The data show that F- I and F-II (0.5 to 10 μg/mL) did not inhibit thrombin when incubated in the absence of AT or HCII. The anticoagulant SPs from green seaweed act via AT and mainly, via HCII, as described for the SPs of the green seaweed Codium adhaerence and Codium divaricatum [18]. Alternatively, they act only via HCII as in the case of the SP of the green seaweeds Caulerpa brachypus and Caulerpa okamurai (HAYAKAWA et al., 2000). Lastly, they can act by both activating serpins and acting directly on thrombin like the Ulva conglobata SP [19]. Thus, the inhibitory effects of F-I and FII on thrombin were assessed in the presence of HCII. The purified SP inhibited thrombin (Figure 2); F-I, at lower concentrations, was more active than F-II. However, neither of the two compounds had any activity greater than 35%.

Fig. 2. Inhibition of thrombin activity by HC-II in the presence of F-I ou F-II. * p<0.05

Figure 3 shows that in the presence of AT, F-II inhibited thrombin activity just as heparin, and starting from 1.0 μg/mL, this effect reached approximately 93% of inhibition. In the case of F-I, a concentration of 0.5 μg/mL showed greater inhibitory effect than heparin (85.3% vs 53.4% of inhibition, respectively), but its inhibitory effect becomes similar to that of heparin starting from 1.0 μg/mL.

Fig. 3. Inhibition of thrombin activity by AT in the presence of F-I ou F-II. * p<0.05

A high anticoagulant activity observed in the aPTT test was mainly due to the inhibition of thrombin by AT. This molecule also inhibits other coagulation factors, thus the possibility that these factors are inhibited by F-I and F-II cannot be discounted, and we intend to evaluate this issue in the future. Our findings show the potential of F-I and F-II as anticoagulant agents since they primarily inhibit antithrombin. We intend to perform in vivo studies to evaluate the anticoagulant activity of F-I and F-II, as well as to determine whether or not these compounds exert any of the collateral effects of heparin. In addition, many studies have indicated the possible importance of anticoagulants in the treatment of different types of tumors [20-21]. The data indicate that tumors induce the formation of

28 tissue factor/factor VIIa complex, which in turn activates factor X and, consequently, thrombin, which leads to thrombus formation. In addition, activated factor X (Xa) and thrombin (IIa) are involved in the proliferation and migration of tumor cells, as well as in the induction of angiogenesis by tumors [22]. Therefore, we also intend to evaluate in the future the antitumor effect of F-I and F-II.

3. Materials and Methods

3.1. Materials

Heparin, N-Benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride chromogenic substrate, heparin cofactor II, monosaccharides, Sephadex G-100, and dextran standards were purchased from Sigma- Aldrich Co. (St. Louis, MO, USA). Cell culture medium components (F-12 medium), trypsin and newborn calf serum (FCS) were obtained from Cultilab (Campinas, SP, Brazil). L-Glutamine, sodium bicarbonate, sodium pyruvate, and phosphate buffered saline (PBS) were purchased from Invitrogen Corporation (Burlington, ON, USA). All other solvents and chemicals were of analytical grade.

3.2. Obtaining of SP-rich extract from the seaweeds

The seaweeds Udotea flabellum (J.Ellis & Solander) M.A. Howe, Caulerpa cupressoides (Vahl) C. Agardh, Caulerpa racemosa (Forsskål) J. Agardh, Caulerpa prolifera (Forsskål) J.V. Lamouroux, Caulerpa sertulariodes (S.G. Gmelin) M.A. Howe, Codium isthmocladum Vickers, Ulva lactuca Linnaeus, Lobophora variegata (J.V. Lamouroux) Womersley ex E.C. Oliveira, Sargassum vulgare C. Agardh, Padina gymnospora (Kützing) Sonder, Spatoglossum schröederi (C. Agardh) Kützing, Dictyota mertensii (Martius) Kützing, Dictyota menstrualis (Hoyt) Schnetter, Hörning & Weber-Peukert, Canistrocarpus cervicornis (Kützing) De Paula & De Clerck, Dictyota ciliolata Sonder ex Kützing, Dictyopteris delicatula J.V. Lamouroux, Acanthophora spicifera (M.Vahl) Børgesen, and Amansia multifida J.V.Lamouroux, were collected from Búzios Beach, Nísia Floresta-RN, Brazil (05° 58’ 23’’ S 35° 04’ 97’ W). The seaweed Gracilaria birdiae E.M. Plastino & E.C. Oliveira, Sargassum filipendula C. Agardh, and Dictyopteris justii J.V. Lamouroux were collected from Rio do Fogo Beach, Rio do Fogo-RN, Brazil (05° 16' 22" S 35° 22' 58" W). The seaweed Gracilaria caudata J. Agardh was collected from Camapum beach, Macau-RN, Brazil (05° 06' 54" S 36° 38' 02" W). The seaweeds were stored in our laboratory and dried at 50 °C under ventilation in an oven, ground in a blender and incubated with ethanol to eliminate lipids and pigments. About 90 g of powdered seaweed was suspended with five volumes of 0.25 M NaCl and the pH was adjusted to 8.0 with NaOH. Next, 900 mg of Prolav 750 (Prozyn Biosolutions, São Paulo, SP, Brazil), a mixture of alkaline proteases, was added for proteolytic digestion. After incubating for 24 h at 60 °C under agitation and periodic pH adjustments, the mixture was filtered through cheesecloth. The filtrates were vacuum dried, resuspended in distilled water, and analyzed.

3.3. Fractionation of SP-rich extract from U. flabellum

The SP-rich extract (2 L) was fractionated by precipitation with acetone as follows: 0.3 volumes of ice-cold acetone was added to the solution under gentle agitation and maintained at 4 °C for 24 h. The precipitate that was formed was collected by centrifugation (10000 x g, 20 min), vacuum dried, resuspended in distilled water, and analyzed. The operation was repeated by adding 0.5, 0.6, 0.7, 1.0, and 2.0 volumes of acetone to the supernatant.

3.5. Fractionation of UF-0.5

The UF-0.5 polysaccharides were separated into two fractions using a 100 kDa centrifugal filter (Amicon® EMD Millipore, Darmstadt, Germany). The retained fraction contained a 130 kDa sulfated galactan F-II. In addition, the filtered fraction was sub-fractionated using 10, 30, and 50 kDa centrifugal filters (Amicon® EMD Millipore, Darmstadt, Germany) to separate different molecules according to

29 their molecular weights. The presence of SPs was detected only in the retained fraction from the 50 kDa centrifugal filter using the Dubois method as described earlier [23].

3.6. Gel permeation chromatography

F-I and F-II were subjected to gel permeation chromatography on Sephadex G-100 (140 x 1 cm) using 0.2 M acetic acid/0.15 M NaCl as an eluent. The elution was monitored for total sugar and metachromasia as described earlier [12]. A set of dextran standards was used to construct the standard curve for the determination of the molecular weights of the polysaccharides.

3.7. Chemical analysis and monosaccharide composition

Sulfate content was determined according to the gelatin-barium method [25], using sodium sulfate (1 mg/mL) as standard and after acid hydrolysis of the polysaccharides (4 M HCl, 100 °C, 6 h). Protein content was measured using Spector’s method [25]. The polysaccharides were hydrolyzed with 0.5, 1, 2, and 4 M HCl for various lengths of time (0.5, 1, 2, and 4 h) at 100 °C. Reducing sugars were determined using the Somogyi-Nelson method [26]. After acid hydrolysis, sugar composition was determined by a LaChrom Elite® HPLC system from VWR-Hitachi (Tokyo, Japan) with a refractive index detector (RI detector model L-2490). A LichroCART® 250-4 column (250 mm x 40 mm) packed with Lichrospher® 100 NH2 (5 µm) was coupled to the system. The samples weighed 0.2 mg and analysis time was 25 minutes. The following sugars were analyzed as references: arabinose, fructose, fucose, galactose, glucose, glucosamine, glucuronic acid, mannose, and xylose.

3.8. APTT test

All Activated Partial Thromboplastin Time (APTT) coagulation assays were performed with a coagulometer as described earlier [27] and measured using citrate-treated normal human plasma. All assays were performed in duplicate and repeated at least three times on different days (n=6).

3.9. AT-mediated thrombin inhibition assay

The thrombin inhibition assay was performed on a 96-well microplate according to the instructions of ACTICHROME® heparin-anti-FIIa Kit (Sekisui Diagnostic, VWR, Radnor, Pennsylvania, EUA). Briefly, 50 μL of antithrombin (AT) was incubated at 37 °C for 2 min in the presence of increasing concentrations of heparin or SPs from U. flabellum, previously diluted in citrated fresh human plasma. Then, 50 μL of bovine thrombin was added to each well, homogenized and incubated at 37 °C for exactly 2 min. Next, 50 μL of the chromogenic substrate was added. The mixture was homogenized and incubated at 37 °C for 2 min. Following incubation, 80 μL 30% acetic acid was added to stop the reaction and absorbance was measured against a corresponding blank at 405 nm.

3.10. HCII-mediated thrombin inhibition assay

The HCII-mediated thrombin inhibition test in the presence of SP was performed in a 96-well microplate with final volume of 100 μL according to Brito et al. [28]. Briefly, 25 μL of the purified SP in 0.02 M Tris/HCl buffer, 0.15 M NaCl (pH 7.4), and 25 μL HCI (70 nM) were incubated for 2 min at 37 ° C. Then 25 μL of thrombin (48 nM) was added and incubated for 1 min at 37 °C. Subsequently, 25 μL of the chromogenic substrate N-benzoyl-Phe-Val-Arg-p-nitroamylidehydrochloride (100 mM) was added and the mixture was incubated for a further 1 min at 37 ° C. Then, 25 μL of 30% acetic acid was added to stop the reaction. The absorbance was read at 405 nm. The blank consisted of a solution with the same reagents, except the substrate was added after the reaction was stopped with 30% acetic acid. To evaluate the total activity of the enzyme (100% enzymatic activity), the test sample was not added.

3.11. Direct thrombin inhibition assay

Direct inhibition of thrombin by SP was determined using the method described above, except HCII was replaced by 0.02 M Tris/HCl buffer, 0.15 M NaCl (pH 7.4).

3.12. Statistical analysis

All data are expressed as mean ± standard deviation (SD) of three observations (n=3). Statistical analysis was done by one-way ANOVA followed by Tukey’s post-test were performed using the Origin program. In all cases, statistical significance was set at p < 0.05.

5. Conclusions Taken together, the results presented in the current study provide strong evidence for the anticoagulant potential of the sulfated galactans from Udotea seaweeds.

Acknowledgments: The authors wish to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico- CNPq (Edital Universal, n° 408369/2016-7), Coordenação de Aperfeiçoamento Pessoal de Nível Superior-CAPES (Higher Level Personal Development Coordination, in loose translation), Programa Ciências do Mar (AUXPE- CIMAR-1956/2014), Programa Nacional de Cooperação Acadêmica (CAPES/PROCAD n° 2965/2014), and Ministério de Ciência, Tecnologia, Inovação e Comércio (MCTI) (the Science and Technology Ministry in Brazil, in loose translation) for the financial support. Hugo Rocha is CNPq fellowship honored researchers (n° 308970/2015- 2). Maxsuell Marques and Rony Viana received a scholarship from CAPES. This research was presented at Programa de Pós-Graduação em Ciências da Saúde at Universidade Federal do Rio Grande do Norte, as part of the M.Sc. thesis of Maxsuel Marques.

Author Contributions: Maxsuell Lucas Mendes Marques and Fernando Bastos Presa prepared the samples and performed the chemical analysis. Rony Lucas Silva Viana, Daniel Lima, Monique Alves, Mariana Santana Santos Pereira Costa, Leandro Silva Costa and Maxsuell Lucas Mendes Marques performed the all the tests. Maxsuell Lucas Mendes Marques and Mariana Santana Santos Pereira Costa analyzed the data. Maxsuell Lucas Mendes Marques and Hugo Alexandre Oliveira Rocha wrote the paper. Edvaldo Silva Trindade and Hugo Alexandre Oliveira Rocha funded and revised the paper

Conflicts of Interest: The authors declare no conflict of interest

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6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES

O grupo de Biotecnologia de Polímeros Naturais (BIOPOL), a qual faço parte desde 2011, realiza uma extensa pesquisa acerca da atividade farmacológica, composição e estrutura molecular de polissacarídeos. Contudo, a inexistência de relatos na literatura sobre os carboidratos da alga verde Udotea flabellum, que está amplamente distribuída no litoral norte riograndense, revelou-se uma área interessante para o desenvolvimento científico até então pouco explorado. A pós-graduação em ciências da saúde possibilitou uma interação com multiprofissionais ainda maior em minha formação, fato esse que contribuiu para guiar de forma mais contundente o cerne do artigo desenvolvido nesse período. Debater e aprender com diferentes mentalidades de pesquisa potencializou a realização desse trabalho. Apesar dos esforços desprendidos, o conhecimento científico precisa ser incessante, deste modo a realização de ensaios in vivo se faz necessário, junto a avaliação angiogênica e trombótica do composto com melhores potenciais de uso. A velocidade, quantidade e a cobrança de coisas que ocorrem durante o mestrado põe em xeque a capacidade da pesquisa e do pesquisador, entretanto para Madre Teresa de Calcutá “Por vezes sentimos que aquilo que fazemos não é, senão uma gota de água no mar. Mas o mar seria menor se lhe faltasse uma gota.”, assim se mostra o fruto deste trabalho, um fragmento intelectual, mas que possa fomentar grandes feitos à sociedade.

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