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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
INTERAÇÃO DE POLISSACARÍDEOS SULFATADOS DA MACROALGA MARINHA Caulerpa cupressoides var. flabellata COM CRISTAIS DE
OXALATO DE CÁLCIO
DAYANNE LOPES GOMES
NATAL/RN 2017
DAYANNE LOPES GOMES
INTERAÇÃO DE POLISSACARÍDEOS SULFATADOS DA MACROALGA MARINHA Caulerpa cupressoides var. flabellata COM CRISTAIS DE
OXALATO DE CÁLCIO
Tese apresentada ao Programa de Pós-
Graduação em Ciências da Saúde da
Universidade Federal do Rio Grande do Norte
como requisito para a obtenção do título de
Doutor em Ciências da Saúde.
Orientador: Prof. Dr. Hugo Alexandre de O. Rocha
NATAL/RN 2017
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
Gomes, Dayanne Lopes.
Interações de polissacarídeos sulfatados da macroalga marinha Caulerpa cupressoides var. flabellata com cristais de oxalato de
cálcio / Dayanne Lopes Gomes. - Natal, 2017. 98f.: il.
Tese (Doutorado) - Programa de Pós-graduação em Ciências da
Saúde. Centro de Ciências da Saúde. Universidade Federal do Rio Grande do Norte.
Orientador: Hugo Alexandre de Oliveira Rocha.
1. Urolitíase - Tese. 2. Alga verde - Tese. 3. Estabilização em COD - Tese. 4. COD haleteres - tese. I. Rocha, Hugo Alexandre
de Oliveira. II. Título.
RN/UF/BS-CCS CDU 616.62-003.7
i
MINISTÉRIO DA EDUCAÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Coordenador do Programa de Pós-Graduação em Ciências da Saúde:
Prof. Dr. Eryvaldo Sócrates Tabosa do Egito
ii
DAYANNE LOPES GOMES
INTERAÇÃO DE POLISSACARÍDEOS SULFATADOS DA MACROALGA MARINHA Caulerpa cupressoides var. flabellata COM CRISTAIS DE
OXALATO DE CÁLCIO
Aprovada em: 20 / 07 / 2017
Banca Examinadora:
Presidente da Banca:
Prof. Dr. Hugo Alexandre de Oliveira Rocha
Membros da Banca
Profa. Dra. Julliane Tamara Araújo de Melo
Profa. Dra. Vanessa de Paula Soares Rachetti
Profa. Dra. Heryka Myrna Maia Ramalho
Profa. Dra. Luciana Nunes Menoli Lanza
iii
Dedico esta obra
A Deus.
Obrigada por me abençoar muito mais do que mereço. Quando deixei de olhar tão
ansiosamente para o que me faltava e passei a olhar com gentileza para o que eu
tinha, descobri que, de verdade, há muito mais a agradecer do que a pedir.
Aos meus pais.
Os maiores incentivadores! Papi Soberano (Zeca) e Mami Poderosa (Célia), os
apelidos retratam a figura grandiosa que vocês representam na minha vida e agradeço
à Deus por tê-los em meu convívio me vendo e me ajudando a trilhar meus caminhos.
Quando falamos de herança, legado, futuro, percebo e reconheço isso porque vocês
me fizeram enxergar que nenhuma herança é capaz de superar o valor da educação.
iv
Dedico esta obra
Aos meus filhos.
Minha força, meu estímulo, minha superação, minha vida. Helena, Heitor e Heloisa:
com vocês aprendo a todo instante, e desses aprendizados o maior deles é saber que
eu sempre posso mais.
A Hugo Rocha (Profi),
Minha enorme gratidão por todos esses anos de orientação. Agradeço pela dedicação,
paciência e, principalmente, pela amizade ao longo desses 11 anos de convivência.
Construímos uma relação de muita confiaça e carinho que sei que jamais se apagará.
“Vivemos com o que recebemos, mas marcamos a vida com o que damos.”
Winston Churchill
v
Agradecimentos especiais
“Eu reconheço que para ti nada é impossível e que nenhum dos teus planos pode ser impedido”. Jó 42:2. Obrigada meu Deus!
Aos meus pais, pelo amor incondicional.
Aos meus filhos, por cada interrupção ao tentar elaborar essa tese. Vocês me fizeram valorizar muito mais essa conquista e também foram o meu refúgio onde eu renovava
minhas forças e retomava a caminhada.
Aos meus irmãos, Dastaev, Dmitryev, Drielle e Dmetryus. Unidos pelo sangue e inseparáveis pelo coração! Sei que posso contar com vocês sempre;
Aos meus compadres e comadres, em especial França por oferecer “amor de graça” a
nós e principalmente a meus filhos, se sacrificando para que eu mantenha minha jornada diária de trabalho. A minha amigan (Jailma) que tanto me ajuda na vida, você é luz, é calmaria, é inspiração. Espero ser para vocês um pouco do que são para mim;
Ao meu esposo Leonardo, pelo companheirismo e paciência. Estando lado a lado, ou
a quilômetros de distância, mas sempre concetados a nossa família pelo amor;
A toda família Rocha de Medeiros, em especial minha sogra Ceiça, que sempre exaltou a importância das conquistas acadêmicas;
Obrigada a todos àqueles do laboratório que fazem ou que já fizeram parte desta
história...
Jailma (Amigan), Mariana, Sara, Karol, Cinthia, Leandro, Diego (Popó), Leonardo Nobre, Ruth, Rafael, Gabriel, Moacir, Talita, Jefferson, Maíra, Polyana, Sr. Paulo, Raniere, Joanna, Letícia, Kaline, Arthur, Vinicius, Max, Rony, Marília, Monique,
Larisse, Mônica, Pablo, Danielle, Almino, Jéssica, Cinthya, Raynara, Adriana, Lucas, Carla, Fernanda (Pôia), Profa. Fabiana, Ana Karina, Ana Karinne (Donana), Fernando,
Ivan, Nednaldo, Duda, Edjane, Valquíria.
Agradeço de forma especial aos amigos do grupo dos cristais: Kerol, Momoxi, Luquete, Pablito, Rafildo. Vocês são top de line!
À UFRN, à Pós-Graduação em Ciências da Saúde e ao Departamento de Bioquímica pela oportunidade de concluir esse curso de Pós-Graduação, assim como as agências
Financiadoras CAPES e CNPq.
A todos os professores do CCS (UFRN), aos coordenadores e equipe do Programa de Pós-Graduação (PPGCSa).
A todos os professores do DBQ (UFRN), em especial, a Profa. Edda Lisboa Leite por ser grande incentivadora e referência pessoal e profissional. Também aos técnicos do
DBQ (UFRN), em especial a Ana Katarina e Francisco Freire pelo apoio direto.
As professoras da banca de qualificação: Profa. Joana Cristina Tavares e Profa. Ariane Lacerda.
Agradeço novamente ao meu orientador Prof. Dr. Hugo Rocha por toda disponibilidade
e tempo dispendido nesse trabalho.
Obrigada a todos!
vi
“Não é sobre chegar no topo do mundo
E saber que venceu
É sobre escalar e sentir
Que o caminho te fortaleceu”.
(Ana Vilela)
vii
RESUMO
A urolitíase afeta aproximadamente 10% da população mundial e está associada
fortemente a formação de cristais de oxalato de cálcio (CaOx). Atualmente não
existe nenhum composto eficiente que possa ser utilizado para prevenir esta
doença. No entanto, os polissacarídeos sulfatados (PS) de algas possuem a
capacidade de alterar a carga superficial dos cristais de CaOx e assim modificar
a dinâmica da cristalização, devido à interação das cargas negativas desse
polímero com a superfície do cristal durante sua síntese. Nestre trabalho foi
verificado o efeito de quatro populações de PS extraídos da alga verde Caulerpa
cupressoides var. flabellata na formação de cristais de CaOx in vitro. Os PS
extraídos foram nomeados de CCB-F0.3, CCB-F0.5, CCB-F1.0 e CCB-F2.0.
Análises de microscopia eletrônica de varredura e de potencial zeta mostraram
que os polissacarídeos extraídos modificam a morfologia, o tamanho e a carga
superficial dos cristais de CaOx formados na presença dos PS. Todos os PS de
C. cupressoides induziram o aumento da quantidade de cristais CaOx formados.
Contudo, com exceção de CCB-F2.0, a presença dos demais PS levou à
formação de cristais de CaOx dihidratados (COD), que são comuns em pessoas
não formadoras de cálculos. Além disso, esses cristais COD apresentaram
morfologia arredondada ou em formato de halteres. As análises de
infravermelho, miscroscopia de fluorescência, citometria de fluxo e a análise de
composição atômica (EDS) permitiram a proposição de um modelo de interação
entre os PS de Caulerpa e os cristais COD. Neste modelo, a distribuição de PS
na estrutura do cristal de CaOx ideal para que haja maior estabilização dos
cristais COD é de 2:1:1 entre a base: ápice: face. Acredita-se que nessa
distribuição os PS consigam evitar a desidratação dos cristais COD, tornando-
os mais estáveis. Este estudo é o primeiro passo para entender as interações
entre PS de C. cupressoides e os cristais de CaOx, que são a principal causa de
cálculos renais.
Palavras-chave. Urolitíase; alga verde; COD halteres; Estabilização em COD.
viii
LISTA DE ABREVIATURAS E SIGLAS
ANOVA Análise de Variância
BIOPOL Laboratório de biotecnologia de polímeros naturais
ºC Graus Celsius
Ca2+ Cálcio
CaOx Oxalato de cálcio
COD Cristal de CaOx Dihidratado
COM Cristal de CaOx Monohidratado
COT Cristal de CaOx Trihidratado
CCB-F0.3 Polissacarídeo precipitado com 0,3 volumes de acetona
CCB-F0.5 Polissacarídeo precipitado com 0,5 volumes de acetona
CCB-F1.0 Polissacarídeo precipitado com 1,0 volumes de acetona
CCB-F2.0 Polissacarídeo precipitado com 2,0 volumes de acetona
ERPS Extrato Rico em Polissacarídeos Sulfatados
EDS Espectroscopia de energia dispersiva
FITC Isotiocianato de fluoresceína
g Grama
GAGs Glicosaminoglicanos
h hora
Hz Hertz de frequência
kV quilovolts
L Litros
M Molar
MEV Microscópio eletrônico de varredura
mg Miligrama
Min. Minutos
mL Mililitros
mM Milimolar
pH Potencial de hidrogênio
IV Infravermelho
MEV Microscopia Eletrônica de Varredura
PBS Tampão salino-fosfato
PS Polissacarídeo (s) Sulfatado (s)
rpm Rotações por minuto
ix
LISTA DE FIGURAS Figura 01. Prevalência Mundial da Urolitíase.............................................................. 13
Figura 02. Alga verde Caulerpa cupressoides var. flabellata em exsicata (Foto:
Mariana Santana).......................................................................................
19
x
SUMÁRIO
RESUMO................................................................................................................................... viii LISTA DE ABREVIATURAS E SIGLAS................................................................................... ix LISTA DE FIGURAS.................................................................................................................. x 1. INTRODUÇÃO....................................................................................................................... 12 2. JUSTIFICATIVA.................................................................................................................... 17 3. OBJETIVOS........................................................................................................................... 18
3.1. GERAL................................................................................................................... 18 3.2. ESPECÍFICOS....................................................................................................... 18
4. MÉTODOS............................................................................................................................. 19 4.1. MATERIAL BIOLÓGICO........................................................................................ 19
4.1.1. Algas....................................................................................................... 19 4.2. EXTRAÇÃO DOS POLISSACARÍDEOS SULFATADOS DA C. cupressoides...... 19
4.2.1. Obtenção de Extratos Ricos em Polissacarídeos Sulfatados (ERPS)... 19 4.2.2. Fracionamento do ERPS com concentrações crescentes de acetona... 20
4.3. SÍNTESE E CARACTERIZAÇÃO DOS CRISTAIS................................................ 21 4.3.1. Ensaio de cristalização de CaOx............................................................ 21 4.3.2. Microscopia Eletrônica de Varredura (MEV) e Espectroscopia de
Energia Dispersiva (EDS).....................................................................
21 4.3.3. Medida do Potencial Zeta (ζ) dos cristais de CaOx............................... 22 4.3.4. Espectroscopia de infravermelho........................................................... 22 4.3.5. Microscopia de fluorescência para análise da morfologia dos cristais
de CaOx................................................................................................
22 4.4. ANÁLISE ESTATÍSTICA........................................................................................ 23
5. ARTIGOS PRODUZIDOS...................................................................................................... 24 5.1. ARTIGO 1 (SUBMETIDO)...................................................................................... 25 5.2. ARTIGO 2............................................................................................................... 57
6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES...................................................................... 76 7. REFERÊNCIAS..................................................................................................................... 78 8. APÊNDICES
8.1. ARTIGO 3............................................................................................................... 82 8.2. ARTIGO 4............................................................................................................... 84 8.3. ARTIGO 5............................................................................................................... 86 8.4. ARTIGO 6............................................................................................................... 88
9. ANEXOS
9.1. NORMAS PARA FORMATAÇÃO DA TESE (CCS)............................................... 91 9.2. NORMAS DA REVISTA PARA SUBMISSÃO (OXIDATIVE MEDICINE AND
CELLULAR LONGEVITY)....................................................................................
94
12
Gomes, D.L. PPGCSA/CCS
1. INTRODUÇÃO
Os rins sãos os órgãos responsáveis pela filtração do plasma, esse é um
processo que ocorre de forma permanente e em grande escala (180 L/dia).
Nesse processo, muitos íons e outras moléculas pequenas vão passar pelos
túbulos renais. Porém, além da alta taxa de filtração, também ocorre uma intensa
reabsorção de moléculas, levando à excreção de apenas algo entre 1 e 1,5 L [1].
Nesse contexto, os íons presentes na urina ficam supersaturados. Alguns
desses íons tendem a interagir com outros e formar sais insolúveis, um processo
comum e natural a todos os indivíduos [2,3]. Esses sais insolúveis vão se
associando de forma lenta, e o produto desta associação vai absorvendo cada
vez mais íons da solução, o que culmina com o surgimento de estruturas
cristalinas em escala nanométrica [4]. A interação entre as cargas dessas
nanoestruturas faz com que elas se atraiam e se agreguem, sendo essa
agregação uma segunda forma de crescimento de cristais, caracterizada pela
rápida deposição de íons e associação a matrizes orgânicas presentes no trato
urinário, fazendo com que a estrutura orgânica/inorgânica formada passe a ser
chamada de cálculo renal ou urolitíase [5].
Do ponto de vista epidemiológico, o cálculo renal atinge cerca de 10% da
população mundial, com maior prevalência nos Estados Unidos [6]. A maior
incidência de cálculo renal ocorre em homens, entre 20 e 60 anos de idade e
tem uma altíssima taxa de recorrência após a primeira crise renal do paciente
[7]. Por acometer uma grande parte da população, a urolitíase gera um grande
impacto na economia e, somente no ano de 2010, os custos associados ao
tratamento hospitalar da urolitíase no sistema público de saúde brasileiro
chegaram a 29,2 milhões no Brasil. Além do alto custo relativo às complicações
que a urolitíase pode gerar, existe o impacto econômico direto por acometer uma
faixa de idade economicamente ativa (20 aos 60 anos) [8].
Do ponto de vista clínico, estes cálculos podem causar dor suprapubica, na
glande do pênis, disuria, hematuria, jato de urina fraco e entrecortado, dentre
outros. Atualmente, o recurso mais utilizado diante de uma crise renal causada
pelo cálculo é a analgesia. Já o tratamento tradicional se baseia na eliminação
de forma espontânea do cálculo, ou ainda procedimentos mais invasivos e com
grau de risco mais crítico, como o cirúrgico tradicional ou bombardeamento a
13
Gomes, D.L. PPGCSA/CCS
laser [9]. Nota-se que mesmo com todos os avanços recentes na medicina ainda
não existe um tratamento realmente eficaz para impedir o surgimento dos
cálculos renais [10].
Figura 01. Prevalência Mundial da Urolitíase. Em vermelho, destacam-se as prevalências de cálculos renais. Em amarelo, destacam-se o crescimento no número de pessoas com cálculos, principalmente de Oxalato de Cálcio [6].
O estágio de formação dos cálculos que consiste na interação inicial dos íons
e na formação das nanoestruturas é chamado de nucleação, pois forma o que
virá a ser um núcleo de crescimento do cristal. Esse núcleo formado serve como
arcabouço para agregação de íons e moléculas e/ou macromoléculas, que vão
levar ao crescimento dos cristais. Existem dois destinos possíveis para esse
núcleo formado: ele pode ser eliminado naturalmente ou pode crescer e agregar
mais cristais, íons e moléculas orgânicas, formando os cálculos renais [11]. Se
ele seguir o segundo caminho, o próximo passo para a formação de cristais é
denominado de agregação, que consiste na união de dois ou mais núcleos entre
si. Essa agregação resulta em cristais grandes e pesados que podem se
precipitar e crescer cada vez mais [12].
Apesar da existência de um mecanismo de formação quase comum a todos
os cálculos, estes podem ser formados a partir de diversos componentes como:
oxalato de cálcio, fosfato de cálcio, ácido úrico, cistina [10] e cristais derivados
de infecção, como estruvita e carbonato de hapatita, além dos cristais mistos
[13]. Apesar dessa grande variedade, mais de 70% dos casos de urolitíase são
formados principalmente por cristais de oxalato de cálcio (CaOx) [14].
14
Gomes, D.L. PPGCSA/CCS
Três tipos de cristais de CaOx podem ser formados no tecido renal, os
cristais de CaOx monohidratados (COM), dihidratados (COD) e trihidratados
(COT) [15]. Devido à supersaturação urinária, naturalmente os três tipos de
cristais de CaOx se formam em todas pessoas. COD e COT são instáveis e tem
uma tendência a se converter em COM, que é o cristal mais estável e o principal
cristal detectado na urina de pacientes formadores de cálculos. Porém, em
pacientes assintomáticos, o cristal mais encontrado na urina é o cristal do tipo
COD [4].
Uma das hipóteses que mostra o papel dos cristais de CaOx na formação
dos cálculos renais relata que o COM possui uma carga positiva, que lhes
permite interagir com moléculas carregadas negativamente encontradas na
superfície celular do epitélio renal, o que leva a sua adesão à superfície da célula
e, por conseguinte, endocitose. A presença desses cristais dentro do citoplasma
celular provoca estresse oxidativo e lesão celular. Um dos resultados desse
processo é a peroxidação lipídica da superfície celular, o que,
consequentemente, provoca a perda de várias moléculas do glicocálice. Este
acontecimento é fundamental para a formação de cálculos renais, pois partes
dessas moléculas do glicocálice que são perdidas tem como função impedir a
interação/adesão de cristais de CaOx com as células. Ou seja, esta perda de
glicocálice permitirá que mais cristais COM se depositem na superfície, não
sejam endocitados e cresçam. Alguns desses cristais vão se combinando entre
si e com outras moléculas para formar cálculos renais, que levam a dano físico
no epitélio e lesão [16, 17].
Uma alternativa interessante para a prevenção da recorrência dos casos de
urolitíase seria o uso de alimentos ou suplementos que pudessem inibir
profilaticamente a formação de cristais [18]. Nessa perspectiva, inserem-se os
glicosaminoglicanos (GAGs), que são polissacarídeos sulfatados (PS)
encontrados nos tecidos, em sua maioria ligados covalentemente a proteínas,
formando os proteoglicanos [19]. No trato urinário, os proteoglicanos da
superfície celular são constituídos de moléculas grandes de GAGs que, por
serem negativamente carregadas, interagem com os cristais de CaOx. Isto
impede a endocitose desses cristais, como também leva à estabilização do
cristal na forma COD. Acredita-se que estes cristais associados aos GAGs
15
Gomes, D.L. PPGCSA/CCS
cresçam até um ponto em que o complexo cristal-fragmento de GAG se solte do
proteoglicano e, consequentemente, da superfície celular. Uma vez solto, o
fragmento do GAG ainda age como estabilizador do cristal, o que justificaria a
presença de cristais COD na urina [20]. Apesar desse papel benéfico, o uso
dessas moléculas como tratamento apresenta uma série de contraindicações,
tais como: i) a sua obtenção a partir de tecido animal, o que implica em alto custo,
baixo rendimento e risco de contaminação do paciente; ii) inúmeras atividades
são exercidas pelos GAGs dentro do organismo, implicando numa grande
chance de ocorrerem efeitos indesejados [19].
Na medicina oriental, algumas algas já são usadas no combate aos cálculos
de CaOx [21]. Essas informações sugerem que as algas marinhas são uma
potencial fonte de moléculas para o tratamento e prevenção de cálculos renais.
Vale salientar que as macroalgas marinhas são fonte de PS e, quando
comparadas a animais, as algas produzem grandes quantidades de PS.
Semelhante aos GAGs, já foi mostrado que PS de algas também tendem a se
associar, por atração eletrostática, às porções positivamente carregadas dos
cristais de CaOx em formação, alterando seu padrão de crescimento [22]. Ao
longo da última década, foi crescente o número de estudos que avaliou atividade
inibidora da formação de cristais de CaOx por PS, especialmente os de algas
marinhas [23].
Tanto Zhang e colaboradores (2012) [24] quanto Melo e colaboradores
(2013) [25], trabalharam com PS de diferentes algas marrons e obtiveram
resultados semelhantes. Zhang e colaboradores trabalharam com um extrato
bruto da alga Sargassum graminifolium e foi capaz de inibir nucleação e
agregação dos cristais de CaOx in vitro. Ainda, os cristais obtidos tinham
morfologia mais arredondada e eram mais estáveis. Já Melo e colaboradores
(2013) trabalharam com 4 polissacarídeos purificados da alga Dictyopteris justii.
Todos eles foram capazes de inibir o crescimento e a agregação de cristais de
CaOx, com destaque para uma glucana sulfatada que também foi capaz de
estabilizar cristais do tipo COD.
Nos dois trabalhos, o mecanismo de ação dos PS foi semelhante ao
observado nos GAGs: interação com o cristal, alteração da carga superficial e
estabilização de formas mais estáveis e menos propensas a causar dano.
16
Gomes, D.L. PPGCSA/CCS
Outro grupo de pesquisadores busca correlacionar esse efeito inibidor da
formação de cristais de CaOx [26, 27] ou de proteção do epitélio renal contra os
danos causados por esses cristais [28] com a quantidade de grupamentos
sulfatos existentes nos PS, já que na maioria dos casos são esses grupamentos
que conferem a carga negativa do polímero. Por exemplo, PS com baixa massa
molecular e diferentes conteúdos de grupamentos sulfato, extraídos de algas
marrons (Undaria pinnatifida, Laminaria japonica, Sargassum fusiforme) e
vermelhas (Porphyra yezoensis, Gracilaria lemaneiformis, Eucheuma) foram
avaliados quando à capacidade de reparar células epiteliais tubulares proximais
de rim humano (HK-2) danificadas com oxalato in vitro. Esse estudo mostrou
uma correlação positiva entre o conteúdo de sulfato e atividade reparadora do
epitélio renal [28]. Essa correlação corrobora com aqueles descritas para PS de
algas que apresentam outras atividades biológicas. Porém, nestes estudos
também é apontado que o fator mais importante para o PS ser bioativo não é só
o fato dele ser sulfatado, mas sim, como estes grupos sulfatos estão distribuídos
ao longo da cadeia molécula de PS [29, 30]. Além disso, os tipos de resíduos
de monossacarídeos que constituem os polímeros parecem também ter
influência sobre as atividades [31].
Ensaios in vivo também mostraram a importância do PS no combate à
urolitíase. Veena e colaboradores trabalhando com PS extraídos da alga marrom
Fucus vesiculosus mostraram que camundongos hiperoxalúricos suplementados
com esses PS tiveram redução do estresse oxidativo em seus organismos,
devido ao aumento da atividade das enzimas antioxidantes e consequente
diminuição da peroxidação lipídica. Este efeito dos polissacarídeos por
conseguinte diminuiu o acúmulo de cristais nos rins dos animais [32]
Ainda não foi proposto um mecanismo completo que explique a ação dos PS
de algas no processo de inibição dos cristais de CaOx [23]. Todavia, essas
moléculas se mostram como uma interessante alternativa na prevenção da
formação de cálculos renais e o entendimento das interações dos PS de algas
com os cristais de CaOx é imprescindível para que eles possam ser utilizados
na terapêutica.
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2. JUSTIFICATIVA
O impacto causado pela urolitíase na sociedade é enorme e cada vez
mais é necessário encontrar fármacos e mecanismos alternativos para que esse
impacto seja reduzido.
Os glicosaminoglicanos (GAGs) são polissacarídeos sulfatados (PS) que
são encontrados na urina e que são tidos como agente inibidores da formação
de cristais de oxalato de cálcio (CaOx). Na década de 1990, vários estudos
avaliaram o efeito de GAGs exógenos na formação de cristais de CaOx [33].
Apesar dos resultados terem sido animadores, um fator importante que levou à
estagnação das pesquisas: a dificuldade de se obter os GAGs em grande
quantidade.
Posteriormente, outros PS passaram a ser estudados com a mesma
finalidade. Dentre eles, PS de algas marinhas (marrons e vermelhas) que,
diferente dos GAGs, são obtidos em grande quantidade. Além disso, muitos
desses PS de algas apresentam atividade antioxidante, que é uma atividade
procurada em agentes utilizados no tratamento de urolitíase [24, 25, 32, 34, 35].
A alga verde Caulerpa cupressoides é encontrada em grande quantidade
no litoral nordestino. Ela sintetiza quatro populações de polissacarídeos, que já
foram caracterizadas pela equipe do Laboratório de Biotecnologia de Polímeros
Naturais-UFRN, sob a coordenação do Prof. Dr. Hugo Rocha, e verificou-se que
estes têm excelente atividade antioxidante em comparação aos PS encontrados
em outras algas do litoral potiguar [30, 36]. Isso associado ao fato de que
nenhuma alga verde teve seus PS avaliados como agentes inibidores da
formação de cristais de CaOx, faz dessa alga uma excelente escolha para a
prospecção de PS com potencial uso no tratamento da urolitiase.
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3. OBJETIVOS
3.1. GERAL
Verificar o efeito dos polissacarídeos sulfatados (PS) da alga verde
Caulerpa cupressoides var. flabellata na formação de cristais de oxalato
de cálcio (CaOx) in vitro.
3.2. ESPECÍFICOS
Extrair PS da alga marinha C. cupressoides utilizando uma metodologia
já desenvolvida pelo nosso grupo de pesquisa;
Analisar o efeito dessas populações de PS sobre a formação de cristais
in vitro;
Caracterizar os cristais de CaOx formados na presença dos PS de C.
cupressoides.
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4. MÉTODOS
4.1. MATERIAL BIOLÓGICO
4.1.1. Algas
A alga marinha verde Caulerpa cupressoides var. flabellata (Figura 02) foi
coletada na Praia de Búzios, município de Nísia Floresta (litoral sul do Rio
Grande do Norte), no mês de março do ano de 2015, em marés baixas entre 0,0
a 0,2 metros a uma temperatura situada entre 28-30°C. As algas foram
recolhidas quando já desprendidas do substrato, mas permanecendo flutuando
nas águas de maré-baixa.
As algas foram acondicionadas em sacos de polietileno e trazidas ao
laboratório no mesmo dia da coleta e, lavadas em água corrente, examinadas
cuidadosamente para remoção de epífitas, inclusões calcárias e sais, sendo
postas para secar em estufa aerada a 45°C. Em seguida, foram trituradas em
liquidificador, pesadas e guardadas em frascos de vidro hermeticamente
fechados.
Figura 02. Alga verde Caulerpa cupressoides var. flabellata em exsicata (Foto: Mariana Santana).
4.2. EXTRAÇÃO DOS POLISSACARÍDEOS SULFATADOS DA C.
cupressoides.
4.2.1. Obtenção de Extratos Ricos em Polissacarídeos Sulfatados (ERPS)
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Gomes, D.L. PPGCSA/CCS
A obtenção dos ERPS da C. cupressoides seguiu o método aplicado pelo
grupo pesquisa que em está inserido esta tese e, mais recentemente, utilizado
por Costa e colaboradores (2010) [30] durante a obtenção de ERPS de algas do
litoral potiguar. Para tanto, as algas secas e pulverizadas foram tratadas com
dois volumes de etanol para despigmentação e delipidação do material. A cada
saturação do álcool, que foi observada visualmente, realizava-se a troca deste
reagente. Posteriormente, o resíduo foi separado do álcool, e colocado para
secar à temperatura ambiente. Após seco, este material despigmentado foi
utilizado para a obtenção dos ERPS. Para a realização dessa etapa, foram
adicionados dois volumes de NaCl a 0,25 M ao pó delipidado (100 g) e o pH
ajustado para 8,0 com NaOH. A esse material foi adicionado a enzima
proteolítica prozima (15 mg/g de pó). Essa suspensão permaneceu em banho-
maria a 60 °C durante um período de 18 h. Depois, foi filtrado e o sobrenadante
submetido a uma centrifugação 10.000 x g por 15 minutos à temperatura de 4
°C. Após a centrifugação, o sobrenadante, que contém os polissacarídeos
solúveis, foi denominado de ERPS, sendo seco à pressão reduzida, triturado,
pesado e guardado para posteriores análises.
4.2.2. Fracionamento do ERPS com concentrações crescentes de acetona
O sobrenadante obtido após proteólise foi fracionado com volumes
crescentes de acetona. Este fracionamento foi realizado de acordo com método
descrito por Costa (2012) [36]. Os valores de acetona adicionados foram
determinados pela turvação da solução, que caracteriza a precipitação de
polissacarídeos devido à adição desse solvente. Inicialmente, foi adicionado 0,3
volumes de acetona, sob agitação leve, volume necessário para que se visualize
uma turvação da solução. Esta foi mantida em repouso a 4 ºC durante 18 h. O
precipitado foi coletado por centrifugação a 8.000 x g por 15 min. a 4 ºC e seco
a pressão reduzida. Em seguida, esse procedimento foi repetido utilizando-se
0.3, 0.5, 1.0 e 2.0 volumes de acetona, obtendo-se as quatro populações
polissacarídicas, denominadas de CCB-F0.3, CCB-F0.5, CCB-F1.0 e CCB-F2.0,
de acordo com o volume de acetona acrescentado.
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4.3. SÍNTESE E CARACTERIZAÇÃO DOS CRISTAIS 4.3.1. Ensaio de cristalização do CaOx
Os cristais de CaOx podem ser formados in vitro a partir de Ca2+ e oxalato.
Para tal, se mistura de cloreto de cálcio (8 mmol/L), oxalato de sódio (1 mmol/L),
cloreto de sódio (200 mmol/L) e acetato de sódio (10 mmol/L), sendo as
concentrações finais desta solução semelhantes às das concentrações
fisiológicas da urina, quando consideradas separadamente. A formação dos
cristais de CaOx foi avaliada a partir da junção dessas soluções formando um
meio superconcentrado favorável à formação de cristais de CaOx. Os cristais
foram formados na presença dos polissacarídeos (250 µg/mL) ou na ausência
(controle) dos polissacarídeos [24]. Após um período de 30 minutos, as soluções
contendo estes cristais foram centrifugadas a 5000 x g e o sobrenatante foi
descartado. O precipitado, composto principalmente por cristais de CaOx, foi
ressuspendido em 0,5 mL de água e uma alíquota de 0,1 mL foi colocada em
lâmina histológica e observada em microscópio ótico (600x) imediatamente após
a ressuspensão. Foram obtidas imagens de 10 campos diferentes aleatórios
para cada lâmina. Em seguida, os diâmetros e tamanhos dos cristais foram
analisados utilizando o software NIS Elements AR Ver4.30.01 DU1 64 bit, ano
de 2014 (Melville, NY, EUA). As conclusões acerca das aferições dos cristais de
CaOx foram obtidas após a realização de experimentos distintos, repetidos
quatro vezes.
4.3.2. Microscopia Eletrônica de Varredura (MEV) e Espectroscopia de
Energia Dispersiva (EDS)
Para se caracterizar a morfologia e composição dos cristais de CaOx na
presença dos polissacarídeos de C. cupressoides, foi realizada a microscopia
eletrônica de varredura MEV (modelo Hitachi Tabletop Microscope TM-3000,
com aceleração de voltagem de 5 kV, frequência de 50/60Hz, magnificação da
imagem de 15 à 30000 vezes), e a Espectroscopia de Energia Dispersiva EDS
(o equipamento utilizado foi Swift ED TM-3000, fabricante Oxford Instruments
detector). As imagens foram geradas com a resolução 1280x960 pixels. Essas
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Gomes, D.L. PPGCSA/CCS
análises foram realizadas no Departamento de Engenharia de Materiais da
Universidade Federal do Rio grande do Norte.
4.3.3. Medida do Potencial Zeta (ζ) dos cristais de CaOx
Após 30 minutos do início da formação dos cristais na presença e na
ausência dos polissacarídeos, as soluções foram centrifugadas (5000 x g). O
sobrenadante foi descartado e o precipitado rico em cristais de CaOx foi
ressuspendido em 1,5 mL de água e avaliado no Zeta Plus® (controle de
temperatura ativo entre -5°C a 110°C, ±0.2°C, 1 a 4 s/ciclo, faixa de pH: 2 a 12,
condutividade: 0 a 7,5 mS/cm, intensidade do campo elétrico: 0 a 3.2 kV/m,) para
obtenção do Potencial Zeta.
4.3.4. Espectroscopia de infravermelho
A espectroscopia de infravermelho foi realizada em espectrômetro Perkin-
Elmer de 4400 a 400 cm-1 no Departamento de Química da Universidade Federal
do Rio grande do Norte. Os cristais de CaOx controle e os cristais de CaOx
formados na presença de cada polissacarídeo de C. cupressoides (~5 mg) foram
analisados após secagem em aparelho de Abdenhalden sob a forma de pastilha
de KBr contendo P2O5 a 60ºC.
4.3.5. Microscopia de fluorescência para análise da morfologia dos cristais
de CaOx
Para melhor compreensão acerca da relação entre a morfologia dos
cristais e disposição dos polissacarídeos sulfatados nos cristais de CaOx, as
populações polissacarídicas foram marcadas com isotiocianato de fluoresceína
(FITC) (Sigma). Resumidamente, 5 mg de cada população foram solubilizadas
numa solução 0,1 M de tampão fosfato (PBS) em pH 7,0 contendo 0,1 mg de
FITC. A solução foi mantida em ambiente com redução de luminosidade, sob
leve agitação, à temperatura ambiente, por 1 hora. Em seguida, o material foi
dialisado contra água deionizada em membranas com poros de 6 kDa de
diâmetro e, posteriormente, liofilizado. As amostras sem polissacarídeos
sulfatados, assim como as amostras de populações marcadas com FITC foram
submetidas a nova produção cristais de CaOx e as lâminas montadas conforme
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Gomes, D.L. PPGCSA/CCS
descrito no item 4.3.1. Para a visualização das imagens (10 campos), foi utilizado
o Microscópio TE-Eclipse 300, Nikon, Melville, NY, USA (Objetiva 60x) e as
imagens foram analisadas através do software NIS Elements AR Ver4.30.01
DU1 64 bit, ano de 2014 (Melville, NY, EUA).
4.4. ANÁLISE ESTATÍSTICA
Todos os dados dos experimentos realizados no item foram expressos
como média ± desvio padrão. Foi utilizado o teste de análise paramétrica de
análise de variância (ANOVA) seguido do teste de Tukey (Nível de significância
de p<0,05) como GraphPad InStat® Software versão 3.05 para Windows 95
(GraphPad Software Incorporation, San Diego, CA, EUA).
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5. ARTIGOS PRODUZIDOS 5.1. Artigo 1 (SUBMETIDO)
Interaction of antioxidant sulfated polysaccharides from green seaweed
Caulerpa cupressoides var. flabellata with calcium oxalate crystals
Periódico: Oxidative Medicine and Cellular Longevity
The reference number for the article is 1284653
Fator de impacto: 4.593
ISSN: 1942-0900 (Online version)
Qualis: Medicina II – A1
5.2. Artigo 2
Methanolic Extracts from Brown Seaweeds Dictyota cilliolata and Dictyota
menstrualis Induce Apoptosis in Human Cervical Adenocarcinoma HeLa
Cells
Periódico: Molecules
Fator de impacto: 2.861
ISSN: 1420-3049 (Online version)
Qualis: Medicina II – B1
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Gomes, D.L. PPGCSA/CCS
5.1 ARTIGO 1
Interaction of antioxidant sulfated polysaccharides from green
seaweed Caulerpa cupressoides var. flabellata with calcium oxalate
crystals
Dayanne L. Gomes1,2, Karoline R. T. Melo1, Moacir Queiroz Fernandes1, Lucas A. N.
C. Batista1, Pablo C. Santos3, Mariana S. S. P. Costa4, Jailma Almeida-Lima1, Rafael
B. G. Câmara1, Hugo A. O. Rocha1*
1 Laboratório de Biotecnologia de Polímeros Naturais (BIOPOL), Departamento de
Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte
(UFRN), Natal, Rio Grande do Norte- RN 59078-970, Brazil.
2 Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Rio
Grande do Norte (UFRN), Natal, Rio Grande do Norte – RN 59078-970, Brazil.
3 Universidade Estadual do Rio Grande do Norte (UERN), Mossoró, Rio Grande do
Norte – RN, 59.610-210, Brazil.
4 Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Norte (IFRN),
Macau, Rio Grande do Norte – RN, 59.500-000, Brazil.
Corresponding author: H. A. O. Rocha
* Email: [email protected]; Tel.: +55-84-32153416 (Branch line: 207).
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Abstract
Urolithiasis affects approximately 10% of the world population and is
strongly associated with calcium oxalate (CaOx) crystals. Currently, there is no
efficient compound that can be used to prevent this disease. However, seaweeds’
sulfated polysaccharides (SPs) have the ability to change the CaOx crystals surface’s
charge and thus modify the crystallization dynamics, due to the interaction of the
negative charges of these polymers with the crystal surface during their synthesis.
We observed that the SPs of C. cupressoides modify the morphology, size and
surface charge of CaOx crystals. Thus, these crystals are similar to those found in
healthy persons. In the presence of SPs, dihydrate CaOx crystals showed rounded
or dumbbell morphology. Infrared analyzes, fluorescence microscopy, flow
cytometry (FITC-conjugated SPs) and atomic composition analysis (EDS) let us
propose the mode of action between the Caulerpa’s SPs and the CaOx crystals. This
study is the first step in understanding the interactions between SPs, which are
promising molecules for the treatment of urolithiasis, and CaOx crystals, which are
the main cause of kidney stones.
Key words. Urolithiasis; Green algae; COD dumbbell; COD stabilization;
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Gomes, D.L. PPGCSA/CCS
1. Introduction
Urinary lithiasis or urolithiasis is a pathophysiological condition resulting from
the formation of kidney stones. The incidence of urinary lithiasis in the world is increasing
rapidly. In the last decade, prevalence doubled from approximately 6 to 11% among men
and 4 to 7% among women, respectively [1]. One of the major problems with urolithiasis
is the risk of developing chronic kidney disease (CKD) and end-stage renal disease
(ESRD), a serious form of CKD [2]. This disease has a high social and economic cost,
since in the USA alone, CKD treatment exceeds $50 billion a year [3]. In England,
according to a recent report published by the National Health Service (NHS) Kidney Care,
costs with CKD case management exceeds those of breast, lung, colon and skin cancers
combined [4].
Urolithiasis can be influenced directly by the types of formed crystals, as each
crystal has its own different ability to bind to the renal epithelium and to initiate the
formation of the stones. Calcium oxalate (CaOx) crystals make up about 70% of urinary
stones [5], and they are found in three different forms: monohydrate (COM), dihydrate
(COD) and trihydrate (COT). The COM crystal is thermodynamically stable, exhibiting
monocyclic geometric morphology (which, in optical microscopy, can be visualized with
a rectangular shape) and showing greater affinity with the renal epithelium. For these
reasons, they are found in kidney stones at a frequency twice as high as that of COD
crystals [6,7]. The COD are commonly found in the urine of asymptomatic persons for
urolithiasis and present a characteristic tetrahedral bipyramidal morphology, which can
be easily visualized by light microscopy [7]. COT crystals show drusiform morphology.
They are unstable, and therefore are rarely found in the urine of patients [8].
The formation of these CaOx crystals is derived from a physico-chemical process
divided into 3 phases: nucleation, aggregation and crystal growth. The preponderant
condition for crystal formation (nucleation) is urinary supersaturation, as due to the high
concentration of ions in a solution, the condensation of these salts occurs forming tiny
crystals, which are called nuclei. The aggregation occurs by the union of one or more
growing nuclei, which form crystals of larger dimension and mass that can precipitate in
the renal epithelium, being able to adhere or to be internalized by cells [9]. The growth
of the kidney stone occurs by aggregation of preformed crystals or by secondary
nucleation of a crystal adhered to the intrarenal structures [10]. The thickness of a crystal
is related to the proportion of its faces. The CaOx crystals have three growth faces, and
each face differs in size and naming according to the type of crystal referred to: faces
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Gomes, D.L. PPGCSA/CCS
(101), (010) and (121) for the COM crystals (Figure 1A); and faces (100), (101) and (011)
for the COD crystals (Figure 1B).
The changes in the CaOx crystal’s face are caused by the other molecules present
in the urinary tract, taking into account the fact that the concentration of ions in healthy
persons and lithogenic patients is practically the same. These molecules, such as citrate,
pyrophosphate, glycosaminoglycans and glycoproteins, present in the renal and urinary
ducts, have the function of stabilizing CaOx crystals, mainly in the COD morphology [11,
12].
Recently, seaweeds sulfated polysaccharides (SPs) also begun to be evaluated for
their anti-lithic capacity. In a recently published review [13], it was verified that these
polymers can inhibit the formation of CaOx renal stone formation in different ways: they
inhibit crystallization (both in the nucleation phase [14,15,16] and in growth phase [16,
17]); they inhibit the aggregation phase [14, 15, 16, 17, 18]; they inhibit the occurrence
of COM crystals and the transformation of COM to COD [14, 16, 17, 18]; and they inhibit
renal tubular cell injury [19,20,21].
The Brazilian coast has a wide diversity of marine seaweed, especially in the
Northeast coast; however, few species had their SPs characterized and evaluated for their
therapeutic potential. The SPs from Caulerpa cupressoides var. flabellata have been
studied by our research group for almost a decade.
Initially, we have shown that SPs-rich extract from C. cupressoides exhibited
several biological activities, including anticoagulant, antiproliferative and antioxidant
activities [22]. Later, four different high molecular weight SPs, named CCB-0.3, CCB-
0.5, CCB-1.0 and CCB2.0 were obtained from this SPs-rich extract. The SPs CCB-F0.3,
CCB-F0.5 exhibited antioxidant activity and significant iron chelating ability [23].
However, the effect of these SPs on the formation of CaOx crystals has not yet been
described. It is noteworthy that, so far, no SP of green seaweed had its action on evaluated
crystal formation. Based on these considerations, the objective of the present study was
to obtain SPs of C. cupressoides (CCB-F0.3, CCB-F0.5, CCB-F1.0 and CCB-F2.0) and
to evaluate their effect on crystallization of CaOx in vitro, as well as from the different
morphological characteristics of the formed CaOx crystals to propose a model of
interaction between the populations of SPs obtained with the CaOx crystals.
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Gomes, D.L. PPGCSA/CCS
2. Materials and Methods
2.1 Sulfated polysaccharides extraction from green seaweed C. cupressoides
After collection, the green seaweed Caulerpa cupressoides var. flabellata
Børgesen was cleaned with running water and oven dehydrated at 45 °C. They were
sprayed and then treated four times with two volumes of ethanol for depigmentation and
delipidation of the material. Two volumes of 0.25 M NaCl were added to the powder
obtained, with the pH being adjusted to 8.0. The proteolytic enzyme maxatase (60 °C, for
18 h) was added to this material. The suspension was then centrifuged at 10.000 g for 20
minutes. The precipitate was discarded and the volume of the supernatant was measured
fractionated with increasing volumes of acetone, obtaining SPs according to a method
established by Costa and collaborators, 2012 [23].
2.2 Chemical and Physicochemical Analysis
The total sugars were determined according to Dubois and collaborators (1956)
by the phenol–H2SO4 reaction [24]. The sulfate content was determined based on the
gelatin/barium method [25]. Protein quantification was determined using Spector’s
method [26]. The SPs were submitted to electrophoresis in order to evaluate the presence
of sulfated polysaccharides and to identify the different populations of these [27].
2.3 Fourier Transformed Infrared (FT-IR) Spectroscopy Analysis
The infrared spectra of the CaOx crystals controls formed after incubation with
the SPs of C. cupressoides were obtained using infrared spectroscopy via Fourier
transform (IRAffinity-1 spectrometer, Shimadzu Corp., Kyoto, Japan) equipped with the
IRsolution 1.20 software. SPs and crystals samples (5 mg) were completely mixed with
dried potassium bromide powder (KBr) and then compressed. The sweep frequency range
was 4000–400 cm−1. Thirty-two scans at a resolution of 4 cm−1 were evaluated and
referenced against air. The Infrared Spectroscopy Analysis was carried out in the
Department of Chemistry of the Federal University of Rio Grande do Norte.
2.4 Calcium Oxalate Crystallization Assay
The CaOx crystals can be formed in vitro from Ca2+ and oxalate to a mixture of
calcium chloride (8 mM/L), sodium oxalate, sodium chloride (200 mM/L) and sodium
acetate (10 mM/L) as final concentrations of this solution from physiological
concentrations of urine. The formation of the CaOx crystals was evaluated in the presence
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Gomes, D.L. PPGCSA/CCS
or absence (control) of the Caulerpa’ polysaccharides [15]. After formation of these, the
solutions consisting of these crystals were centrifuged at 5.000 x g and the supernatant
was discarded. The precipitate is composed mainly of crystals of CaOx, and was
resuspended in 0.5 mL of water, and a solution of 0.1 mL was placed on a histological
slide and observed under an optical microscope (600x). Images of 10 different fields were
obtained for each slide, then the crystal diameters and sizes were analyzed using the NIS
Elements AR Ver4.30.01 DU1 64-bit software, year 2014 (Melville, NY, USA). The
conclusions about the measurements of the CaOx analyzes were obtained after a trial of
distinct experiments, being repeated four times.
2.5 Scanning electron microscopy (SEM) and Dispersive Energy Spectroscopy
(EDS)
To observe the superstructure and composition of generated crystals in the
presence of the SPs of C. cupressoides, a scanning electron microscopy (Hitachi Tabletop
Microscope TM-3000 model, with 5 kV voltage acceleration, 50/60Hz of frequency,
image magnification from 15 to 30000) and dispersive energy spectroscopy (Swift ED
TM-3000, Oxford Instruments detector) images were generated with 1280x960 pixels
resolution. The Scanning Electron Microscopy was carried out in the Department of
Materials Engineering of the Federal University of Rio Grande do Norte.
2.6 Zeta Potential (ζ) Measurements
After 30 minutes of crystal formation in the presence and absence of the
polysaccharides [15], the solutions were centrifuged (5000 x g). The crystal concentrate
was then suspended in 1.5 mL of water, and the zeta potential of the ζ samples was
obtained using a Zeta Plus® analyzer (active temperature control between -5°C at 110°C,
±0.2°C, 1 at 4 s/cycle, pH range: 2 at 12, conductivity: 0 at 7.5 mS/cm, intensity of the
electric field: 0 at 3.2 kV/m), Brookhaven instruments, Holtsville, NY, USA.
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Gomes, D.L. PPGCSA/CCS
2.7 Light microscopy and fluorescence microscopy for the analysis of CaOx crystal
morphology
To better understand the morphology and arrangement of the SPs in the CaOx
crystals, polysaccharide was labeled with fluorescein isothiocyanate (FITC). 5 mg of each
polysaccharide was added to 0.1 mL of phosphate buffer (PBS) at pH 7.0 containing 0.1
mg FITC. The solution was kept in an environment with reduced brightness, at room
temperature for 1 hour. The material was then labeled with deionized water in membranes
with pores of 6 kDa, and then lyophilized. Samples without SPs, as well as samples of
FITC-labeled polysaccharides were not subjected to new production of CaOx crystals and
slides, assembled according to item 2.4. Images were captured from different fields under
a fluorescent microscope (TE-Eclipse 300, Nikon, Melville, NY, USA). We performed
three different experiments.
2.8 Statistical Analysis
All of the data are expressed as the mean ± standard deviation (n = 3). The
ANOVA test was performed to check the difference between results. The Student–
Newman–Keuls test (p < 0.05) was used to solve similarities found by ANOVA. All tests
were performed in GraphPad Prism 5 (GraphPad Softwares, La Jolla, CA, USA).
3. Results and Discussion
3.1 C. cupressoides’s sulfated polysaccharides extraction, chemical and
physicochemical analysis
In a previous study [23], our group extracted four populations of sulfated
polysaccharides from the C. cupressoides seaweed. These populations were named as
CCB-F0.3, CCB-F0.5, CCB-F1.0, and CCB-F2.0, respectively. In this work, using the
same previously described methodology, we also obtained four polysaccharide
populations from C. cupressoides. These samples were subjected to agarose gel
electrophoresis in PDA buffer. In figure 2 we showed an electrophoresis slide stained
with toluidine blue, the toluidine blue has affinity for SPs. We can see the four populations
stained with toluidine blue, indicating that they are constituted of SPs. Different
electrophoretic mobility can also be verified: CCB-F0.3, CCB-F0.5 are polysaccharides
of low mobility in comparison with the others and CCB-F1.0 presented intermediate
mobility, whereas CCB-F2.0 was the population with higher mobility. By comparing our
32
Gomes, D.L. PPGCSA/CCS
data with the electrophoresis slide presented by Costa and
colleague [23], we can confirm the identity of the polysaccharides obtained.
As observed previously [23], in all extracted SPs, the protein contamination was
below 0.1% (Table 1), confirming the efficacy and reproducibility of the extraction
methodology.
Table 1 revealed a higher sulfate/sugar ratio for CCB-F0.3 (~1.10) and the lowest
ratio for CCB-F2.0 (~ 0.72). These values differ from those previously observed [23];
these authors found CCB-F0.5 with higher sulfate/sugar ratio and CCB-F1.0 with the
lowest ratio of the four fractions. These data were not surprising, since many authors
report changes in the chemical compositions of polysaccharides extracted from the same
species of seaweed when collected at different sites [28, 29]. However, seaweed, both in
Costa and colleges [23] as in here, were collected at the same site (6 ° 1'8.19 "S - 35 °
6'33.40" W), which discards this possibility, even though they were collected in different
years, which takes seaweed to be exposed to different environmental conditions, leading
to changes in the SPs composition. This effect has already been reported by different
authors, but these variations are not homogeneous; each species of seaweed responds
differently to the environmental changes in the collection site, like the green seaweed
Ulva fasciata’s SPs varied in yield, monosaccharide composition and sulfate amount [30];
on the other hand, Delesseria sanguínea’s SPs’ monosaccharide composition and sulfate
content have not been affected by the seasonality [31].
In order to evaluate whether the chemical composition differences of SPs in this
study interfered with the biological activity described earlier [23], we choose the
antioxidant test of iron chelation to verify if the activity of SPs obtained here was similar
to that previously described. As seen in Table 1, the iron chelating activity of the samples
varied from 34 ± 2% (CCB-F1.0) to 53 ± 1% (CCB-F0.5). These values are similar to
those observed by Costa and colleagues [23] and indicates that, despite the differences
between sulfate/sugar ratios found in this study and previously published [23], we believe
that these variations promote only subtle differences in polysaccharide structure, and
these differences are not sufficient to affect the activities of SPs of C. cupressoides. This
led us to continue our studies with the obtained polysaccharides.
33
Gomes, D.L. PPGCSA/CCS
3.2 Effect of sulfated polysaccharides C. cupressoides on the formation of calcium
oxalate crystals
It is possible to mimic the formation of these crystals in vitro [15] and to evaluate
the effect of compounds on the formation of these crystals. Based on light field optical
microscopy, we can infer the effect of SPs on the formation of crystals by quantification
of each type of crystal. The data obtained are summarized in Table 2.
When we analyze the data showed in the table, we can see that the presence of
polysaccharides increased the amount of crystals formed. In the presence of CCB-F0.3,
the amount of crystals increased 12-fold, followed by CCB-F1.0, which increased by
approximately 9-fold. However, despite the increase in the amount of crystals, the size of
the crystals formed when treated with C. cupressoides seaweed SPs was reduced about
four-fold on average, reaching, in some cases, about 1 µm after treatment (Table 2). The
CCB-F0.3 again stood out by decreasing 7 times the size of COM and 5 times the size of
COD.
Wesson and collaborators [32] proposed that anionic compounds tend to increase
the number of crystals and decrease their size since these negatively charged compounds
interact more with the rich calcium faces of both COM and COD crystals, blocking their
growth [32]. In addition, the anionic compounds induce repulsions between the formed
nuclei (the nano/micro crystals), preventing the aggregation process, as they are
associated with the crystals. These two factors prevent the formation of larger
crystals. With respect to the large number of crystals formed in the presence of anionic
compounds, we believe that as the ions are not completely consumed during nuclei
formation, these are available for the formation of new nuclei. These factors together
justify the observation of the large number of small crystals formed in the presence of C.
cupressoides SPs.
Also, in Table 2, we can see that all obtained SPs (except CCB-F2.0) induced the
formation of an amount of COD-type larger than COM-type, and, again, CCB-F0.3 was
highlighted, as in the presence of CCB-F0.3, the number of crystals was 209 ± 12.3 units
of COD type, much higher than the number of COM type (48 ± 10.9 units), making a
ratio of approximately 4 COD for each COM.
It is important to emphasize that COM-type crystals, when formed in vivo, can be
concentrated in the renal tubular fluid and interact with kidney tubular epithelial cells,
giving them enough time to grow on the cell surface or to aggregate mutually so as to
form large crystals, which finally leads to the formation of kidney stones. On the other
34
Gomes, D.L. PPGCSA/CCS
hand, COD crystals, due to their morphology, have an area of contact with the minimal
renal epithelium and, therefore, bind the cell membrane in a smaller amount [33].
Other SPs of different seaweeds showed the ability to inhibit the formation of
COM crystals or to stabilize the dihydrate form of the CaOx crystal (COD). For example,
Ouyang and colleagues [14] worked with the edible seaweed Laminaria japonica and
found that its native and modified SPs induced the formation of only COD crystals
[14]. However, SPs from Dictiopteris justii are able to inhibit the formation of CaOx
crystals. In addition, its sulfated glucan was also able to stabilize CaOx crystals in the
COD form [16]. There is no hypothesis that explains how SPs stabilize CODs. However,
studies with carboxylated polymers with the same net charge showed that they did not
influence the COD stability in the same way, and it was proposed that the distribution of
the charges around the molecule was a more important factor than the charge alone during
this process of stabilization [34]. So, this should probably be an important factor for SPs
as well.
3.3 Morphology of the crystals formed in the presence of Caulerpa polysaccharides
For a more detailed analysis of the morphological characteristics of the formed
crystals, the images were obtained by scanning electron microscopy (SEM) and were
analyzed subsequently. The results are summarized in Figure 3.
The images of Figure 3 corroborate with the results of Table 2, showing that there
is an increase in the amount of formed crystals, but decreasing their sizes. Still in
agreement with Table 2, there are more COD-type crystals in the crystal images formed
after incubation with CCB-F0.3, CCB-F0.5 and CCB-F1.0 (Fig. 3C to 3G) when
compared to Control crystals (Figures 3A and 3B). The crystals that were formed without
the incubation with the SPs (control) had the morphological characteristic of COM and
COD, marked by isotropic growth on all the faces of the crystals. Moreover, we observed
control only in the crystals of the COT-type (arrow tipped diamond, Figure 3B), i.e., all
treatments with seaweed SPs C. cupressoides inhibited the formation of this crystal.
Regarding the COD crystals, after treatment, they showed a more rounded shape
(without well-defined tips) than the control group. CCB-F0.3 (Figure 3C) induces the
formation of nearly round, spherical crystals with the tiny (100) faces, evidenced by the
arrow in Figure 3D. The CCB-F2.0 and CCB-F1.0 polysaccharides induce the formation
of COD, which assumes the tetragonal bipyramid shape, but with the tips slightly
rounded. CCB-F0.5 induces the formation of crystals with tetragonal bipyramid structures
35
Gomes, D.L. PPGCSA/CCS
with thicker (100) faces (white arrow with arrowhead, Figure 3E and 3F); besides, these
crystals have rounded edges when compared to COD formed in the control.
Still regarding COD crystals, we also observed that both CCB-F0.5 and CCB-F1.0
induces the formation of a mixture of COD crystals, which, in addition to the already
described types, also forms dumbbell-shaped crystals. These crystals showed two
hemispheres connected with a rod (Figure 3F). COD crystals with traditional bipyramidal
structure have the face (101) as the dominant face. According to Thomas and colleagues
[35], the COD crystals only take the form of dumbbells when the ratio between faces
{100} and {101} is greater than 1, that is, the face (100) grows much more than the face
(101). These authors also demonstrated that the presence of polyacrylate (PAA) also
promotes the formation of COD crystals in the form of dumbbells. This indicates that
COD crystals in the form of dumbbells can be formed in the presence of some anionic
compounds. However, we still do not know what SP’ structural features are required for
this to occur.
Regarding COM crystals, when formed after treatment with C. cupressoides's
SPs, they were smaller and had structure with the faces {100} dominating. There was an
increase in growth in the direction [010] rather than growth in the direction [100]. This
makes COM have edges and tips with less sharp angles, as they are shaped like an ellipse
(oval in the form of a plaque). Thus, the morphological characteristic assumed by COM
crystals after incubation with C. cupressoides's SPs is advantageous, as while leaving the
edges of their faces with a slight angle (rounded), the possible interaction of these crystals
in renal epithelium can be decreased, which will consequently favor its passage in the
urinary tract, thus reducing the formation of kidney stones. In agreement with our
hypothesis, there are studies that analyzed the crystals of CaOx contained in the urine of
lithogenic patients, and they verified that the COM crystals had edges and tips with sharp
angles and concluded that these are important factors for the anchorage of these crystals
in the renal epithelium [33, 36].
3.4 FT–IR spectrum analyses
The COM and COD crystal FT-IR spectra are showed in Figure 4. We can observe
that the main difference between these spectra is the region between 3040 and 3500 cm-
1. While in COM crystal spectra, there are at least five prominent bands (Figure 4) in this
same region. In COD crystal spectra, there is only one great intensity band around 3485
cm-1 (Figure 4 – COD spectra). Other typical signal of COM crystal were observed around
36
Gomes, D.L. PPGCSA/CCS
947, 885 and 663 cm-1 (Figure 4 – COM spectra), whereas characteristic COD signals
were observed at 916 and 609 cm-1. Both spectra are similar with those previously
described for COM and COD crystals [37, 38].
Since we observed that more COD crystals were formed in the presence of CCB-
F03 and more COM crystals were formed in the presence of CCB-F2.0, these samples
were chosen to perform infrared analysis. In these spectra of the crystals formed in the
presence of CCB-F0.3 and CCB-F2.0, there are bands around 1232-1256 cm-1, which
indicate asymmetric vibration S=O [39], and the bands around 1150 e 1025-1033 cm-1
are indicative of vibration associated to C-O-S-O [40]. These bands were also observed
in the spectra of the crystals obtained in the presence of the other SPs (data not shown)
and confirm the presence of SPs CCB-F0.3, CCB-F0.5, CCB-F1.0 and CCB-F2.0 in
CaOx crystals.
Spectra analysis also allows noticing that, in the presence of CCB-F0.3, there is
a predominant formation of COD crystals, since a single band was observed in the region
between 3040 and 3500 cm-1. However, in the CCB-F2.0 spectra, it was possible to
observe multiple bands in this region, indicating that SPs are mainly complexed with
COM crystals. These data thus corroborate with those obtained by microscopic analyzes.
3.5 Zeta potential
The zeta potential (ζ) of crystals formed in the presence of C. cupressoides’ SPs
was measured in order to verify if changes in the number and/or morphology of crystals
were associated with changes in the surface charge of these crystals. The results are shown
in Table 3.
The ζ mean value of untreated CaOx crystals was + 8.85 ± 3.30 mV. This positive
profile of crystal charge surface can be mainly related with the presence of calcium ions
in crystal structure. All SPs decreased the zeta potential of CaOx crystals, ranging from -
25.82 ± 6.36 mV in the presence of CCB-F0.3 to -68.70 ± 12.01 mV in the presence of
CCB-F2.0.
An interesting fact is that the increase of crystals ζ did not correspond to the
sulfate/sugar ratio (Table 1), that is, polysaccharide with high amounts of sulfate groups
did not promote formation of crystals with lower ζ value. Other authors have already
noted this fact [15, 16], and it is proposed that SP associated with crystal tends to assume
a conformation that allows it to have higher or lower exposition of its charged groups.
Therefore, it is a situation where, in a negatively charged polysaccharide, it may assume
37
Gomes, D.L. PPGCSA/CCS
a conformation in which its charges are not so exposed in the crystal surface, resulting in
ζ of the crystal being closer to zero.
The ζ increases with the presence of SPs of C. cupressoides, which explains part
of the formation of many small crystals. The elevated negative charge on crystal surface
would lead to repulsion of other crystals and would block crystal aggregation and
growth. This interference in crystal aggregation/growth has also been observed by other
authors while working with other seaweeds SPs [15, 16]. In those works, the authors also
report formation of many small crystals and elevated crystal negative charge.
Zeta potential analyzes provide us great indications that SPs interact with CaOx
crystals structure. However, there are no studies that actually prove direct binding
between SPs from seaweed and CaOx crystals. Despite, this connection is well described
for other molecules that also have negative charges in their structure, such as polyacrylate
[35], osteopontin [41, 42], glycosaminoglycans [43], and citrate [44], for example.
Therefore, it can be confirmed, based on the data presented here and compared with those
in the literature, that SPs seaweeds are able to alter the surface charge of CaOx crystals
and thereby modify their crystallization dynamics.
3.6 Fluorescence and flow cytometry analyzes of CaOx crystals and FITC-labeled
SPs.
In another set of experiments, the SPs were covalently conjugated to the FITC, as
described in Methods (item 2.7), and used as fluorescent probes with goal to observe
binding of SPs to the formed crystals. To this end, FITC-labeled polysaccharides were
incubated with the supersaturated solutions inducing the formation of CaOx crystals, and
the crystals resulting from this incubation were analyzed by flow cytometry and
fluorescence microscopy. As control, crystals formed in the presence of fluorescently
unlabeled SPs were used.
As expected, crystals formed in the presence of unfluorescent SPs were not
detected by flow cytometry. However, using this technique, we verified that 90% of
crystals formed in the presence of fluorescent SPs showed positive FITC signal.
When those crystals were analyzed by fluorescence microscopy, we noticed that
they appear marked by FITC (green) in almost all of their totality (Figure 5); besides, we
were able to differentiate formed COM and COD crystals. These data give evidence that
SPs are interacting with entire crystalline network, probably with calcium present in these
crystals.
38
Gomes, D.L. PPGCSA/CCS
3.7 Stabilization of COD crystals
The data presented so far showed that, with the exception of CCB-F2.0, all other
SPs of C. cupressoides induce higher amount of COD crystals in comparison to the
positive control. In addition, SPs gave COD crystals’ higher stability. This behavior has
already been observed by Escobar and colleagues [45], in the presence of other sulfated
(dermatan sulfate, oversulfated heparin and keratan sulfate) and phosphorylated
(phosphorylated chitosan) polysaccharides. Interestingly, there was a greater formation
of COM crystals when these authors used desulfated dermatan, chitosan and hyaluronic
acid (carboxylated, but not sulfated polysaccharide). These results show the importance
of sulfate groups in polysaccharides for the stabilization of COD crystals [45].
However, not every sulfated polysaccharide promotes stabilization of COD
crystals, as shown by Melo and colleagues [16] and also by our results, since CCB-2.0
induces more COM formation. This shows that the SP’s COD stabilizing effect is not
merely a charge effect, but it also depends on how charges are distributed across the
polysaccharide chain. Moreover, these data show that COD formed in the presence of the
different C. cupressoides’s SPs may take different forms, which seems to indicate that
SPs can stabilize COD in different ways, perhaps because they associate with crystals in
different faces.
In order to confirm this hypothesis, the surfaces of the COD crystals obtained after
the incubation with the SPs studied here had their atomic composition characterized
through the spectroscopic chemical microanalysis of X-rays by energy dispersion (EDS).
The sulfur atoms were quantified at different points of the crystals: apex, face and base
(Figure 6A). The sulfur found on the surface of the crystals should represent the sulfate
(SO42-) groups of SPs, since CaOx is composed only of calcium, carbon and oxygen, so
some considerations have been made.
The results from analyzes are summarized in the table of Figure 6B. We observed
in this table that three situations occur in the sulfur distribution: the first occurs in the
crystals formed with CCB-F0.3 and CCB-F2.0, where there is twice the amount of sulfur
in the region of the base than in the other portions; The second occurs with the crystals
formed with CCB-F0.5, where there are almost ten times more sulfur at the base of these
crystals than at the apex or face; The last situation is when there is a different distribution
at the base, face and apex, with the amount of sulfur at the base being twice as large as
the apex, as is the case with CCB-F1.0.
39
Gomes, D.L. PPGCSA/CCS
We found no other articles that have done this type of analysis; therefore, it was
not possible to compare our results with those of other authors. However, some
considerations have been made.
Is it necessary for the SPs to be distributed differently throughout the crystal so
that there is a greater stabilization of the COD crystals? It does not seem so, since CCB-
F1.0 has this type of distribution, but the COD:COM ratio obtained with this sample was
only 2: 1 (Table 2).
The other SPs were more concentrated on the base. However, there is a subtle
relationship between the amount of sulfur that should be at the base, apex and face. If
there is a lot of sulfur in the base (about ten times more) than in the other points, as
occurred with the presence of CCB-F0.5, there is stabilization of COD, but with only a
COD: COM ratio of 3: 1 (Table 2). The ideal distribution for COD stabilization seems to
be that observed with CCB-F0.3, since the COD: COM ratio was 5:1. The CCB-F0.3
concentrates more on the base, but its amount is only twice of that observed in the other
points; in addition, the amount of this SP at the apex and face are similar. This distribution
profile was also observed with CCB-F2.0. However, we found that the crystals formed
with this polysaccharide have twenty times less sulfur than the crystals formed with CCB-
F0.3.
4. Conclusions
Four sulfated polysaccharides were extracted from the seaweed Caulerpa
cupressoides and were named CCB-F0.3; CCB-F0.5; CCB-F1.0; and CCB-F2.0. These
SPs interact in vitro with calcium oxalate crystals, making their surface’s more negative.
They also induce the decrease in the size and number of formed crystals. This effect did
not depend on the amount of sulfate groups present in SPs. With the exception of CCB-
F2.0, SPs induce the formation of a greater amount of COD crystals compared to the
control group, with CCB-F0.3 being the most efficient. This occurred because CCB-F0.3
was distributed in the base:apex:face of the crystal in a ratio of 2:1:1. We believe that this
balance/proportion between SPs at the interaction points is crucial to avoid dehydration
of the COD crystal to COM, which guarantees their stability.
Acknowledgements
The authors wish to thank Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Coordenação de Aperfeiçoamento Pessoal de Nível Superior
40
Gomes, D.L. PPGCSA/CCS
(CAPES) and also Ministério de Ciência, Tecnologia, Inovações e Comunicações
(MCTI) for the financial support. Hugo Rocha is an honored CNPq fellowship researcher.
Dayanne Lopes Gomes, Karoline Rachel Theodósio Melo and Moacir Queiroz Fernandes
Neto have a Ph.D. scholarship from CAPES. The Infrared Spectroscopy Analysis was
carried out in the Department of Chemistry of the Federal University of Rio Grande do
Norte, and the Scanning Electron Microscopy was carried out in the Department of
Materials Engineering of the Federal University of Rio Grande do Norte. The authors
would like to thank and acknowledge the permission to use the facilities of these
laboratories. 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 Ph.D. thesis of
Dayanne Lopes Gomes.
Conflicts of Interest
The authors declare no conflict of interest.
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[33] J. Y. He, S. P. Deng, and J. M. Ouyang, “Morphology, particle size distribution,
aggregation, and crystal phase of nanocrystallites in the urine of healthy persons and
lithogenic patients”, IEEE Transactions on Nanobioscience, vol. 9, no. 2, pp. 156–
163, 2010.
[34] X. Sheng, T. Jung, J. A. Wesson, and M. D. Ward, “Adhesion at calcium oxalate
crystal surfaces and the effect of urinary constituents”, Proceedings of the National
Academy of Sciences of the United States of America, vol. 102, no. 2, pp. 267–272,
2005.
[35] A. Thomas, E. Rosseeva, O. Hochrein, et al., “Mimicking the growth of a pathologic
biomineral: shape development and structures of calcium oxalate dihydrate in the
presence of polyacrylic acid”, Chemistry–A European Journal, vol. 18, no. 13, pp.
4000–4009, 2012.
[36] C. Y. Duan, Z. Y. Xia, G. N. Zhang, B. S. Gui, J. F. Xue, and J. M. Ouyang, “Changes
in urinary nanocrystallites in calcium oxalate stone formers before and after
potassium citrate intake”, International Journal of Nanomedicine, vol. 8, no. 1, pp.
909–918, 2013.
[37] H. Peng, J. M. Ouyang, X. Q. Yao, and R. E. Yang, “Interaction between submicron
COD crystals and renal epithelial cells”, International Journal of Nanomedicine, vol.
7, pp. 4727–4737, 2012.
[38] X. Y. Sun, J. M. Ouyang, A. J. Liu, Y. M. Ding, and Q. Z. Gan, “Preparation,
characterization, and in vitro cytotoxicity of COM and COD crystals with various
sizes”, Materials Science and Engineering: C, vol. 57, pp. 147–156, 2015.
[39] T. M. A. Silva, L. G. Alves, K. C. S. Queiroz, et al., “Partial characterization and
anticoagulant activity of a heterofucan from the brown seaweed Padina
gymnospora”, Brazilian Journal of Medical and Biological Research, vol. 38, no. 4,
pp. 523–533, 2005.
[40] Y. Cao and I. Ikeda, “Antioxidant activity and antitumor activity (in vitro) of
xyloglucan selenious ester and sulfated xyloglucan”, International journal of
biological macromolecules, vol. 45, no. 3, pp. 231–235, 2009.
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[41] B. Grohe, J. O'young, D. Ionescu, et al., “Control of calcium oxalate crystal growth
by face-specific adsorption of an osteopontin phosphopeptide”, Journal of the
American Chemical Society, vol. 129, no. 48, pp. 14946–14951, 2007.
[42] Y-C. Chien, D. L. Masica, J. J. Gray, S. Nguyen, H. Vali, and M. D. McKee,
“Modulation of calcium oxalate dihydrate growth by selective crystal-face binding
of phosphorylated osteopontin and polyaspartate peptide showing occlusion by
sectoral (compositional) zoning”, Journal of Biological Chemistry, vol. 284, no. 35,
pp. 23491–23501, 2009.
[43] S. E. R. Hernandez and N. H. Leeuw, “Effect of Chondroitin 4-Sulfate on the Growth
and Morphology of Calcium Oxalate Monohydrate: A Molecular Dynamics Study”,
Crystal Growth & Design, vol. 15, no. 9, pp. 4438−4447, 2015.
[44] S. R. Qiu, A. Wierzbicki, C. A. Orme, et al., “Molecular modulation of calcium
oxalate crystallization by osteopontin and citrate”, Proceedings of the National
Academy of Sciences, vol. 101, no. 7, pp. 1811−1815, 2004.
[45] C. Escobar, A. Neira-Carrillo, M. S. Fernández, and J. L. Arias, “Role of sulfated
macromolecules in urinary stone formation”, Biomineralization: From Paleontology
to Materials Science, pp. 343–358, 2007.
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Tables
Table 1. Chemical characterization and Chelating ability of SPs from C. cupressoides. D-
galactose and sodium sulfate were used as standards for sugar and sulfate quantification,
respectively. The concentration of each SP used to perform the iron chelation test was 1.5
mg/mL. Data are expressed as the mean of three determinations ± standard deviation.
Different letters indicate significant difference for the same amount of SPs (p <0.05).
Polysaccharide (SO4)/total sugar
(%/%) Protein
(%) Chelating ability
(%) CCB-F0.3 1.10 ± 0.02 a
0.06 ± 0.01 a 36 ± 4 a
CCB-F0.5 0.86 ± 0.03 b
0.05 ± 0.01 b 53 ± 1 b
CCB-F1.0 0.85 ± 0.02 b 0.11 ± 0.02 c 34 ± 2 a
CCB-F2.0 0.72 ± 0.01 c 0.11 ± 0.01 c 13 ± 4 c
a, b, c Different letters indicate a significant difference between the SPs of C. cupressoides (p <0.05)
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Table 2. Number and average size of the crystals formed by treatment with sulfated
polysaccharides from C. cupressoides. COM - monohydrate calcium oxalate crystals;
COD - dihydrate calcium oxalate crystals; COT - trihydrate calcium oxalate crystals.
COTs were only found in the control (1 ± 0.9 units).
Total amount
of crystals
(units)
COM (units) COM size
(µm)
COD
(units)
COD size
(µm)
Control CaOx 21 ± 4.4 13 ± 4.1 11.9 ± 0.16 7 ± 2.0 12.8 ± 0.89
CaOx + CCB-F0.3 257 ± 4.2 48 ± 10.9 1.7 ± 0.08 209 ± 12.3 2.55 ± 0.19
CaOx + CCB-F0.5 77 ± 8.3 20 ± 6.9 4.2 ± 0.64 58 ± 9.8 4.9 ± 0.12
CaOx + CCB-F1.0 184 ± 10.8 66 ± 9.1 4.4 ± 0.29 118 ± 9.8 4.7 ± 0.79
CaOx + CCB-F2.0 32 ± 9.4 23 ± 7.3 4.8 ± 0.96 9 ± 3.3 6.2 ± 0.83
.
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Table 3. Zeta potential of crystals formed in the presence of polysaccharide fractions of
C. cupressoides seaweed at 25 °C.
a, b, c, d Different letters indicate a significant difference (p <0.05) in Zeta Potential between CaOx formed
with the presence or absence of polysaccharides.
ζ (mV)
CaOx + 8.85 ± 3.30a
CaOx + CCB-F0.3 - 25.82 ± 6.36b
CaOx+ CCB-F0.5 - 43.87 ± 8.63c
CaOx + CCB-F1.0 - 51.50 ± 2.14c
CaOx + CCB-F2.0 - 68.70 ± 12.01d
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Figure Legends
Figure 1. Representation of the faces of the calcium oxalate crystals. (A) Crystals COM;
(B) Crystals COD. The numbers presented between "parentheses" represent each face of
a crystal; The numbers displayed between “brackets” represent the direction of growth of
each face of a crystal; And, the numbers presented between "braces" represent the set of
same faces of a same crystal.
Figure 2. Electrophoretic mobility of the sulfated polysaccharides from C. cupressoides
seaweed. 50 μg of the Caulerpa SPs were applied on agarose gel prepared with 1,3-
diaminopropane-acetate buffer 0.05M, pH 9.0, and then subjected to electrophoresis at
90 V/cm for 60 min. The gel was maintained in 0.1% cetyltrimethylammonium bromide
for 2 h, dried and subsequently stained with 0.1% toluidine blue (in 50% ethanol and 1%
acetic acid in water) for 15 min. The gel was unstained with the same staining solution
without the dye.
Figure 3. Scanned Electron micrographs of the crystals formed morphotypes after
incubation with polysaccharides of green seaweed C. cupressoides. The CaOx crystals
were formed in a stable CaOx solution target (1 mM) in the absence (A and B images)
and in the presence of SPs C. cupressoides (0.25 mg/mL). (C and D) CCB-F0.3; (E and
F) CCB-F0.5; (G) CCB-F1.0; (H) CCB-F2.0. Shows the COM shape;
Shows the COD form and shows the COT form.
Figure 4. Infrared spectrum of COM, COD and CaOx crystals formed after incubation
with Caulerpa SPs. FT-IR with main signals found in infrared spectra of CaOx crystals
formed in the presence and absence (control) of CCB-F0.3 and CCB-F2.0. Signs in red
represent the unique peaks of COM; Signals in blue represent the unique COD peaks;
Pink signs represent the fashion hybrids for COM and COD; Signs in green represent the
sugar or sulfate peaks.
Figure 5. Fluorescent crystal analyzes by flow cytometry and fluorescence microscopy.
(A) Fluorescence quantification of crystals labeled by fluorescent SPs and detected by
flow cytometry, expressed as percentage; (B) Comparison between crystals in light field
microscopy (non-fluorescent), observed in the upper line, and the SPs-FITC-conjugated
crystals (fluorescent in green), observed in the bottom line. The assays were performed
twice (n = 3).
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Figure 6. Crystals surfaces’s atomic composition characterization by EDS. (A)
Representation of selected sites for quantification of sulfur atoms (apex, face and base);
(B) Percentage of sulfur distribution in different parts of CaOx and CaOx treated with
polysaccharide; (C) Calcium (red dots) and oxygen (blue dots) marking in CaOx with the
presence of CCB-F0.5; (D) Increase in sulfur marking (green dots) in CaOx with the
presence of CCB-F0.5.
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Figures
Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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5.2. ARTIGO 2
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6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES
A busca para elucidar um possível mecanismo de interação com os PS e
os cristais de CaOx nos fez recorrer a disversas técnicas, o que conferiu um
caráter interdisciplinar ao trabalho. Realizamos testes em departamentos de
diversas áreas da UFRN (Química, Engenharia de Materiais e Instituto do
Cérebro). Portanto, o desenvolvimento deste estudo me proporcionou a
oportunidade de interagir com diversos ramos da pesquisa científica e considero
esse fato imprescindível não só para conseguir boas publicações como também,
e principalmente, para formação profissional e científica.
Ao longo do desenvolvimento desta pesquisa, o cronograma de atividades
seguiu dentro dos padrões esperados e todas as dificuldades encontradas foram
superadas. No 3º mês após o ingresso no programa fiquei grávida. Porém, isso
não interferiu no cumprimento das disciplinas. O fato de já estar inserida no grupo
da pesquisa há 11 anos facilitou para que eu dinamizasse a pesquisa no retorno
da licença maternidade. Não posso deixar de citar os colaboradores do próprio
laboratório (BIOPOL/UFRN), que de forma séria porém ao mesmo tempo
bastante descontraída nos ajudam a vencer os obstáculos no decorrer dos anos.
É importante ressaltar que durante esse período além da condução da
minha pesquisa de doutorado fiz algumas orientações às pesquisas dos alunos
de iniciação científica e também colaborações com outros projetos de mestrado
e doutorado. Alguns desses trabalhos já foram publicados, outros estão em
processo de construção e submissão/aprovação dos artigos. Dos já publicados
nos últimos quatro anos estão a produção de 6 artigos publicados em revista
científicas, sendo um como primeira autora, dois como segunda autora e os
demais dividindo co-autoria. As revistas foram: International Journal of Biological
Macromolecules, Evidence-Based Complementary and Alternative Medicine,
Marine Drugs, Molecules e Artificial Organs. Além disso, também fiz parte de 5
(cinco) bancas examinadora de trabalhos de conclusão de curso (monografia).
Durante esse processo de formação também participei de alguns
Congressos, Simpósios e Encontros científicos internacionais e nacionais como
autora ou em co-autoria de 18 resumos científicos, como por exemplo nos 42º e
45º Annual Meeting of the Brazilian Society for Biochemistry and Molecular
Biology (SBBq), 2013 e 2016. Destaco a minha participação no IV CURSO DE
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FÉRIAS DE MORFOLOGIA: UM ELO ENTRE OS DISCENTES DA FACISA E
DO ENSINO MÉDIO ministrando a palestra “Mitos e Verdades sobre Cálculos
Renais” no ano de 2016. Essa iniciativa de passar o meu conhecimento científico
para a comunidade foi bastante enriquecedor e retrata o real papel da
Universidade.
Como projeto para o futuro, planejamos continuar com os estudos na
mesma linha de pesquisa, em um futuro pós-doutorado para finalizar alguns
questionamentos levantados ao longo da pesquisa que prometem excelentes
resultados.
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7. REFERÊNCIAS
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12. Ogawa Y, Miyazato T, Hatano T. Oxalate and Urinary Stones. World J
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13. Bichler K H, Eipper E, Naber K, Braun V, Zimmermann R, Lahme S.
Urinary infection stones. Int J Antimicrob Agents. 2002; 19 (6): 488-498.
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YQ, An RH. Oxalate Impairs Aminophospholipid Translocase Activity In
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16. Lieske J C, Deganello S, Toback F G. Cell-crystal interactions and kidney
stone formation. Nephron, 1999; 81 (1): 8-17.
17. Yuen J W, Gohel M D, Poon N W, Shum D K, Tam P C, Au D W. The initial
and subsequent inflammatory events during calcium oxalate lithiasis. Clin
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18. Strohmaier W L. Economics of stone disease/treatment. Arab J Urol.
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20. Lulich, J. P. Effects of a urolith prevention diet on urine compositions of
glycosaminoglycans, Tamm-Horsfall glycoprotein, and nephrocalcin in
cats with calcium oxalate urolithiasis. Am J Vet Res. 2012; 73 (3): 447-
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21. Tsai C H, Chen Y C, Chen L D, Pan T C, Ho C Y, Lai M T, Tsai F J, Chen
W C. A traditional Chinese herbal antilithic formula, Wulingsan, effectively
prevents the renal deposition of calcium oxalate crystal in ethylene glycol-
fed rats. Urol Res. 2008; 36 (1): 17-24.
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E S, Mittler S, Goldberg H A, Hunter G K. Crystallization of Calcium
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Crystal Interactions. Langmuir. 2009; 25 (19): 11635-11646.
23. Bhadja P, Lunagariya J, Ouyang J-M. Seaweed sulphated polysaccharide
as an inhibitor of calcium oxalate renal stone formation. J Funct Foods.
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24. Zhang C Y, Wu W H, Wang J, Lan M B. Antioxidant Properties of
Polysaccharide from the Brown Seaweed Sargassum graminifolium
(Turn.), and Its Effects on Calcium Oxalate Crystallization, Mar Drugs.
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25. Melo K R, Camara R B, Queiroz M F, Vidal A A, Lima C R, Melo-Silveira
R F, Almeida-Lima J, Rocha H A. Evaluation of Sulfated Polysaccharides
from the Brown Seaweed Dictyopteris Justii as Antioxidant Agents and as
Inhibitors of the Formation of Calcium Oxalate Crystals. Molecules. 2013;
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26. Ouyang J-M, Wang M, Lu P, Tan J. Degradation of sulfated
polysaccharide extracted from algal Laminaria japonica and its modulation
on calcium oxalate crystallization. Mater Sci Eng C. 2010; 30: 1022-1029.
27. Ouyang J M, Wu X M. Morphological and phase changes in calcium
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Eucheuma striatum. Chem Letters. 2005; 34 (9): 1296-1297.
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Polysaccharides with Different Contents of Sulfate Group and Molecular
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29. Haroun-Bouhedja F, Ellouali M, Sinquin C, Boisson-Vidal C. Relationship
between sulfate groups and biological activities of fucans. Thromb Res.
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30. Costa L S, Fidelis G P, Cordeiro S L, Oliveira R M, Sabry D A, Câmara R B, Nobre L T, Costa M S, Almeida-Lima J, Farias E H, Leite E L, Rocha H A. Biological activities of sulfated polysaccharides from tropical seaweeds. Biomed Pharmacother. 2010; 64: 21-28.
31. Ngo D H, Kim S K. Sulfated polysaccharides as bioactive agents from marine algae Int J Biol Macromol. 2013 ; 62 : 70-75.
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sulfated polysaccharides from edible seaweed Fucus vesiculosus in
experimental hyperoxaluria. Food Chem, 2007; 100 (4): 1552-1559.
33. Cao L C, Deng G, Boevé E R, Romijn J C, de Bruijn W C, Verkoelen C F,
Schröder F H. Does urinary oxalate interfere with the inhibitory role of
glycosaminoglycans and semisynthetic sulfated polysaccharides in
calcium oxalate crystallization? Eur Urol.1997; 31 (4): 485-492.
34. Zhang C Y, Kong T, Wu W H, Lan M B. The protection of polysaccharide
from the brown seaweed Sargassum graminifolium against ethylene
glycol-induced mitochondrial damage. Mar Drugs, 2013; 11: 870-880.
35. Veena C K, Josephine A, Preetha S P, Varalakshmi P, Sundarapandiyan
R. Renal peroxidative changes mediated by oxalate: The protective role of
fucoidan. Life Sci. 2006; 79: 1789-1795.
36. Costa M S S P, Costa L S, Cordeiro S L, Almeida-Lima J, Dantas-Santos
N, Magalhães K D, Sabry D A, Albuquerque I R L, Pereira M R, Leite E L,
Rocha H A O. Evaluating the possible anticoagulant and antioxidant
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cupressoides var. flabellata. J Appl Phycol. 2012; 24 (5): 1159-1167.
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APÊNDICES
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8.1. ARTIGO 3
Baccharis trimera (Less.) DC Exhibits an Anti-Adipogenic Effect by
Inhibiting the Expression of Proteins Involved in Adipocyte Differentiation
Periódico: Molecules
Fator de impacto: 2.861
ISSN: 1420-3049 (Online version)
Qualis: Medicina II – B1
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8.2. ARTIGO 4
Bothrops jararaca and Bothrops erythromelas Snake Venoms Promote
Cell Cycle Arrest and Induce Apoptosis via the Mitochondrial
Depolarization of Cervical Cancer Cells
Periódico: Evidence-Based Complementary and Alternative Medicine (Print)
Fator de impacto: 1.931
ISSN: 1741-427X (Printed version)
Qualis: Medicina II – B1
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8.3. ARTIGO 5
Fucan-coated silver nanoparticles synthesized by a green method induce
human renal adenocarcinoma cell death
Periódico: International Journal of Biological Macromolecules
Fator de impacto: 3.138
ISSN: 0141-8130 (Online version)
Qualis: Medicina II – A2
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8.4 ARTIGO 6
Evaluation of Anti-Nociceptive and Anti-Inflammatory Activities of a
Heterofucan from Dictyota menstrualis
Periódico: Marine Drugs
Fator de impacto: 3.503
ISSN: 1660-3397
Qualis: Medicina II – A2
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ANEXOS
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9.1. NORMAS PARA FORMATAÇÃO DA TESE (CCS)
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9.2. NORMAS DA REVISTA PARA SUBMISSÃO (OXIDATIVE MEDICINE AND
CELLULAR LONGEVITY)
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