caracterização dos polissacarídeos extracelulares ... · cromatografia em coluna de gel de...
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CCaarraacctteerriizzaaççããoo ddooss ppoolliissssaaccaarrííddeeooss eexxttrraacceelluullaarreess
pprroodduuzziiddooss ppoorr CChhllaammyyddoommoonnaass ccff..
ppuummiilliioonniiffoorrmmiiss ((CChhllaammyyddoopphhyycceeaaee)):: iiddeennttiiffiiccaaççããoo
ddee ffrraaççõõeess ccoomm ppootteenncciiaall bbiiooaattiivviiddaaddee..
Lucas da Silva Maria
Orientador: Dr. Danilo Giroldo
Rio Grande 2012
Universidade Federal do Rio Grande Instituto de Ciências Biológicas Pós-graduação em Biologia de
Ambientes Aquáticos Continentais
CCaarraacctteerriizzaaççããoo ddooss ppoolliissssaaccaarrííddeeooss eexxttrraacceelluullaarreess pprroodduuzziiddooss ppoorr
CChhllaammyyddoommoonnaass ccff.. ppuummiilliioonniiffoorrmmiiss ((CChhllaammyyddoopphhyycceeaaee))::
iiddeennttiiffiiccaaççããoo ddee ffrraaççõõeess ccoomm ppootteenncciiaall bbiiooaattiivviiddaaddee..
Aluno: Lucas da Silva Maria
Orientador: Dr. Danilo Giroldo
Rio Grande 2012
Universidade Federal do Rio Grande Instituto de Ciências Biológicas
Pós-graduação em Biologia de Ambientes Aquáticos Continentais
Dissertação apresentada ao Programa
de Pós-graduação em Biologia de
Ambientes Aquáticos Continentais como
requisito parcial para a obtenção do
título de Mestre em Biologia de
Ambientes Aquáticos Continentais.
AGRADECIMENTOS
• Ao Prof. Dr. Danilo Giroldo, pelo inspirador exemplo de professor e pesquisador e
pela diligente e paciente orientação nesses últimos 6 anos de trabalho e convivência.
• À Universidade Federal do Rio Grande (FURG) e ao Programa de Pós
Graduação em Biologia de Ambientes Aquáticos Continentais (PPG-BAC), palcos não
só de uma formação acadêmica de excelência, mas também de lembranças que ficarão
para a vida toda.
• Ao CNPQ, pelo apoio financeiro na realização desse projeto.
• À professora Dra. Mariângela Menezes, do Museu Nacional do Rio de Janeiro,
pela identificação de Chlamydomonas cf. pumilioniformis (prestes a ser confirmada).
• Ao técnico Sr. Vanderlen e ao Prof. Dr. João Sarkis Yunes, do Instituto de
Oceanografia da FURG, pelo auxílio com a obtenção das frações extracelulares de
Chlamydomonas cf. pumilioniformis. E aos técnicos Cláudio e Leonardo, do Laboratório
de Limnologia do Instituto de Ciências Biológicas (ICB) da FURG, pela disponibilidade e
apoio em diversas atividades.
• Ao Prof. Dr. Adalto Bianchini, do ICB, pelo apoio logístico na montagem das
colunas de cromatografia.
• À Profa. Dra. Ana Baisch, à Profa. Dra. Cristiana Dora e à Msc. Marilia Garcez, do
Laboratório de Ensaios Farmacológicos e Toxicológicos (ICB), pelo contagiante
entusiasmo pela ciência, pela troca de conhecimentos e pelo apoio logístico e técnico na
obtenção das frações polissacarídicas.
• Aos componentes da banca examinadora dessa dissertação, pelas valiosas
considerações, sugestões e correções.
• Aos colegas e amigos de laboratório, especialmente Miriam, Pablo, Savênia,
Bianca e Kelen, pelas discussões científicas e filosóficas e pelas prazerosas
comemorações gastronômicas de cada conquista acadêmica.
• Ao Alan, ao Moisés, à Priscila e à Daiane, pela amizade e pelo incrível poder de
motivação.
• A toda minha família, especialmente aos meus pais, Sr. Silvio e Lúcia Maria, que
com tanto apoio, motivação e sacrifícios também merecem seus títulos acadêmicos.
• À minha noiva Raquelita, por seu amor, carinho, compreensão e encorajamento.
E pela história que ainda vamos escrever juntos.
• A Deus, por criar as microalgas, esses reatores bioquímicos fotossintetizantes
com informação suficiente para a realização de infinitas pós-graduações.
RESUMO
As microalgas são organismos diversificados não apenas em sua variedade de
grupos taxonômicos e habitats, mas também em sua capacidade de produzir um amplo
espectro de compostos com potencial interesse industrial e farmacológico, dentre os
quais se destacam os polissacarídeos. Este estudo objetiva caracterizar os
polissacarídeos extracelulares produzidos por Chlamydomonas cf. pumilioniformis
(Chlamydophyceae) em cultivos estanque axênicos. A espécie envolvida neste trabalho
foi isolada por micromanipulação ao microscópio do lago raso subtropical Polegar,
localizado no Campus Carreiros da Universidade Federal do Rio Grande (FURG) e está
sendo mantida na Coleção de Culturas de Microalgas Dulcícolas da FURG (CCMD-
FURG). Frações dos polissacarídeos extracelulares produzidos pela alga foram obtidas
por cromatografia em coluna de gel de troca iônica (Sepharose DEAE “Fast Flow”) e
exclusão por massa molecular (Sephacryl S400) e, posteriormente, foram
caracterizadas por cromatografia iônica de alto desempenho com detecção por
amperometria pulsada (HPIC-PAD). Foram isoladas seis frações com diferentes
composições monossacarídicas, sendo que em apenas uma, rica em glucose, ribose e
frutose, a arabinose não foi detectada. Nas outras cinco frações predominaram
arabinose, galactose e glucose em diferentes proporções. Considerando as seis
frações isoladas, também foram detectadas menores proporções de fucose, ramnose,
N-acetil-galactosamina, N-acetil-glucosamina, manose, xilose, ácido galacturônico e
ácido glucurônico, sendo que os dois últimos somaram quase 20% em uma das
frações. O elevado teor de arabinose e galactose observado na maior parte das frações
aproximam a composição destes polissacarídeos às pectinas de plantas vasculares,
que possuem reconhecidos efeitos biológicos, como antiinflamatório, imunoestimulador
e vasodilatador. Esses resultados reforçam a relação entre os efeitos biológicos de
extratos brutos de C. pumilioniormis demonstrados em experimentos anteriores
realizados com camundongos e a composição das frações polissacarídicas produzidas
por essa alga.
Palavras-chave: Volvocales, carboidratos, cromatografia, arabinose.
ABSTRACT
Microalgae are very diverse organisms not only in the variety of taxonomic
groups and habitats, but also in their ability of producing a wide range of compounds
with potential industrial and pharmacological applications, including polysaccharides.
This study aimed to characterize the extracellular polysaccharides produced by
Chlamydomonas cf. pumilioniformis (Chlamydophyceae) in axenic batch cultures. It was
isolated from a small subtropical shallow lake and has been maintained in the
Freshwater Microalgae Culture Collection of FURG (CCMD-FURG). Polysaccharide
fractions were obtained by ion exchange (DEAE Sepharose "Fast Flow") and size
exclusion chromatography (Sephacryl S400) and they were characterized by high
performance ion chromatography with pulsed amperometric detection (HPIC-PAD). We
isolated six fractions and only one of them, rich in glucose, ribose and fructose, was not
composed of arabinose. The five other fractions were mainly composed of arabinose,
galactose and glucose in different proportions. Considering all fractions, we also found
minor proportions of fucose, rhamnose, N-acetyl-galactosamine, N-acetyl-glucosamine,
mannose, xylose, galacturonic acid and glucuronic acid, and the two last are
responsible for 20% of one of the fractions. The high content of arabinose and
galactose make such polysaccharide similar to pectins from higher plants, which have a
well known biological effect e.g. anti-inflammatory, immunostimulatory and vasodilator.
Our results reinforce the significance of such polysaccharide composition to previously
verified biological activity in mice of C. pumilioniormis crude extract.
Key-words: Volvocales, carbohydrates, chromatography, arabinose.
SUMÁRIO
1) INTRODUÇÃO GERAL ............................................................................................................ 9
2) REFERÊNCIAS BIBLIOGRÁFICAS ...................................................................................... 12
3) MANUSCRITO SUBMETIDO À REVISTA JOURNAL OF APLLIED PHYCOLOGY ...... 16
4) INSTRUÇÕES DA REVISTA JOURNAL OF APPLIED PHYCOLOGY .............................. 29
5) ANEXO: MEIO WC ................................................................................................................. 34
LISTA DE FIGURAS
Figura 1. Frações do polissacarídeo extracelular de C. pumilioniformis isoladas pela
cromatografia de troca iônica (Sepharose “Fast Flow” Pharmacia®) e exclusão por massa
molecular (Sephacryl S400 Pharmacia®).
Figura 2. Cromatografia em coluna de gel de troca iônica DEAE-Sepharose “Fast Flow”
Pharmacia® dos polissacarídeos excretados por C. pumilioniformis, eluídos com água destilada e
NaCl 0,5 M. O fluxo foi de 0,3 mL min-1 em uma coluna de 150 mL. Ve/Vt = Volume
Eluído/Volume Total da Coluna.
Figura 3. Cromatografia em coluna de gel de exclusão por massa molecular Sephacryl S400
Pharmacia® dos polissacarídeos ácidos (0,5 M) e neutros (H2O) excretados por C.
pumilioniformis, eluídos com butanol 2%. O fluxo foi de 0,3 mL min-1 em uma coluna de 120
mL. Ve/Vt = Volume Eluído/Volume Total da Coluna. A seta vazada indica a saída do
polissacarídeo “blue dextran” (2x106 D), ao passo que a seta preenchida indica a saída do
dextran obtido de Leuconopsis spp, de massa molecular de 5x105 D.
Figura 4. Cromatogramas da análise por HPIC-PAD da composição monossacarídica das
Frações A (neutra de maior massa molecular), B (neutra de massa molecular intermediária) e C
(neutra de menor massa molecular).
Figura 5. Cromatogramas da análise por HPIC- PAD da composição monossacarídica das
Frações D (ácida de maior massa molecular), E (ácida de massa molecular intermediária) e E
(ácida de menor massa molecular).
Figura 6. Composição monossacarídica das frações polissacarídicas neutras (A, B e C) e ácidas
(D, E e F) de C. pumilioniformis. Fucose (Fuc), Ramnose (Ram), N-Acetilgalactosamina
(NAcGal), Arabinose (Ara), N-Acetilglicosamina (NAcGli), Galactose (Gal), Glicose (Gli),
Manose (Man), Xilose (Xil), Frutose (Fru), Ribose (Rib), Ácido Galacturônico (AGal) e Ácido
Glicurônico (AGli).
LISTA DE TABELAS
Tabela 1. Distribuição dos polissacarídeos extracelulares produzidos por C. pumilioniformis nas
frações isoladas pela cromatografia de troca iônica (Sepharose “Fast Flow” Pharmacia®) e
exclusão por massa molecular (Sephacryl S400 Pharmacia®) em massa (mg) e porcentuais (%).
1) INTRODUÇÃO GERAL
As microalgas são seres vivos muito diversificados que ocupam praticamente todos os tipos
de ambientes da biosfera, tais como água doce, salgada, gelo, solos, rochas e cascas de árvores,
ocorrendo nos ambientes mais extremos como regiões polares e desérticas graças às suas eficientes
adaptações morfofisiológicas. Do ponto de vista filogenético, incluem desde linhagens muito
primitivas até grupos modernos, configurando uma definição mais funcional do que propriamente
taxonômica (Van den Hoek et al 1995). Esta variedade de linhagens evolutivas, formas e
mecanismos fisiológicos faz das microalgas um grupo produtor de uma grande variedade de
compostos químicos com potencial aplicação nas indústrias alimentícia, cosmética, na produção de
energia e como fonte de substâncias com potencial aplicação farmacêutica (Olaizola 2003).
Aliada à potencialidade das microalgas como produtoras de compostos de interesse
industrial, observa-se a facilidade no isolamento de muitos destes organismos de ambientes naturais
e na sua manutenção em condições controladas de cultivo em coleções de culturas (Lourenço 2006).
Uma vez em cultivo, é possível testar condições ambientais que favoreçam o crescimento e a
produção de determinadas substâncias, bem como avançar no conhecimento genético da produção
de tais compostos (De Phillipis et al 2001). Embora o cultivo de microalgas seja uma atividade de
crescente interesse dos pesquisadores desde meados do século XX e o potencial da aplicação
biotecnológica das microalgas seja largamente reconhecido, uma porcentagem muito pequena da
biodiversidade brasileira deste grupo está sendo mantida em coleções de cultivo, já que a
quantidade de coleções bem estabelecidas no Brasil é ainda baixa (Lourenço e Vieira 2004,
Borowitzka 1995). O pouco conhecimento sobre a potencialidade da biodiversidade nacional de
microalgas dificulta a prospecção de espécies que detenham propriedades de interesse e é um
estímulo para a ampliação dos estudos de caracterização bioquímica de microalgas isoladas em
sistemas aquáticos brasileiros, com vistas à identificação de compostos com potencial aplicação
farmacológica e industrial, o que identifica as algas como um grande reservatório inexplorado de
aplicações biotecnológicas (Piccardi et al 2000).
Dentre os metabólitos produzidos pelas microalgas, destacam-se quantitativamente e
qualitativamente os polissacarídeos tanto intra como extracelulares. São compostos importantes do
ponto de vista quantitativo, pois correspondem de 40 a 90% dos compostos orgânicos produzidos
por estes organismos e qualitativamente representam um amplo espectro de compostos com
composições e massas moleculares diferenciadas (Myklestad 1995). De modo geral, podem-se
separar os polissacarídeos das microalgas em três grandes grupos: reserva, estruturais e
extracelulares. Os polissacarídeos de reserva são os principais polissacarídeos intracelulares,
geralmente formados por glucanos homogêneos, como o amido e suas variações das cianobactérias
e rodófitas, bem como a crisolaminarina (Percival 1979) típica das algas heterocontes (diatomáceas,
10
crisofíceas, xantofíceas e outras) e o paramilo das euglenofíceas. Os polissacarídeos estruturais,
presentes principalmente nas paredes celulares, podem variar bastante em termos de composição
dependendo do grupo taxonômico, como xilanos, mananos, ramnanos (Carlberg e Percival 1977) e
glicoproteínas variadas (Morita et al 1999), sendo um importante critério na definição de filos,
classes e ordens de microalgas (Reviers 2006). Já os polissacarídeos extracelulares, que também
podem ser bastante representativos do ponto de vista quantitativo (Paulsen e Vieira 1994), possuem
uma composição muito variável podendo formar compostos homogêneos como fucanos (Giroldo e
Vieira 2002), mananos (Vieira et al 2006), arabinogalactanos (Kiemle et al 2007), bem como
heteropolissacarídeos com 5 ou mais componentes (Giroldo e Vieira 2005). Este grupo de açúcares
é menos conhecido, tanto em termos de composição e estrutura, como de suas funções biológicas
para as próprias microalgas (Giroldo et al 2007). Embora os polissacarídeos extracelulares possam
ser produzidos especificamente por exudação (Giroldo e Vieira 2005), o conjunto dos
polissacarídeos encontrados externamente às células das microalgas sofre influência dos
polissacarídeos estruturais, que se desprendem de paredes de células vivas e mortas, bem como
daqueles de reserva que ganham o meio extracelular com a lise de células mortas.
A grande variedade de frações com diferentes massas moleculares e composições variadas
produzidas pelas inúmeras espécies de microalgas ainda não estudadas indica uma significativa
possibilidade de descoberta de compostos bioativos. Dentre os polissacarídeos frequentemente
observados em microalgas, e que potencialmente teriam atividade biológica, destacam-se os
fucanos, arabinanos, arabinogalactanos e ramnogalacturanos (Paulsen 2001).
Os fucanos são polissacarídeos ricos em fucose e têm um largo espectro de atividade
biológica, incluindo efeito anticoagulante, anti-inflamatório, imunoestimulador, anti-viral, anti-
tumoral e anti-metástase (Cumashi et al 2007). As principais fontes destes compostos são as
macroalgas pardas (Classe Phaeophyceae), particularmente das Ordens Laminariales e Fucales,
produtoras de um tipo de fucano conhecido como fucoidan, que além da alta proporção de fucose,
conta também com alto grau de sulfatação. Também diversos grupos de invertebrados marinhos,
como as holotúrias, produzem fucanos bioativos, porém em quantidades bem inferiores às
macroalgas (Motohiro 1960). Além destes organismos, diversas espécies de microalgas produzem
fucanos com potencial bioatividade, porém os testes para comprovar a real atividade destes
compostos isolados de microalgas são ainda bastante raros. Uma cepa da cianobactéria
Synechocystis aquatilis apresentou uma produção de um polissacarídeo sulfatado rico em fucose e
arabinose com atividade anticoagulante (Volk et al 2006). Cryptomonas obovata, Cryptomonas
tetrapyrenoidosa (Cryptophyceae), Thalassiosira duostra (Bacillariophyceae), Staurastrum
orbiculare (Zygnematophyceae) e Onychonema sp. também são exemplos de microalgas dulcícolas
produtoras de polissacarídeos ricos em fucose com potencial bioatividade ainda inexplorada
11
(Giroldo e Vieira 2002, Giroldo 2003, Giroldo et al 2005 a e b, Maria et al 2011).
Os arabinanos, arabinogalactanos e ramnogalacturanos são freqüentemente associados a
uma classe de compostos denominada pectinas, que podem variar muito em termos de composição,
mas têm como característica marcante a presença de arabinose, galactose, ramnose e ácido
galacturônico. Tanto pectinas neutras, como os arabinanos e os arabinogalactanos, quanto ácidas,
como os ramnogalacturanos, têm reconhecida bioatividade, principalmente anti-inflamatória e
imunoestimuladora (Paulsen 2001). Estes compostos são muito freqüentes em plantas de uso
medicinal, sendo um dos alvos principais da farmacognosia. Plantas vasculares como Biophytum
petersianum e Panax ginseng têm sido usadas em tratamentos relacionados ao sistema imune, assim
como dores nas juntas, inflamações e malária, e sua atividade tem sido relacionada a polissacarídeos
pécticos, como ramnogalacturanos e arabinogalactanos (Inngjerdingen et al 2008, Yu et al 2010).
Diversas espécies de algas, principalmente as mais próximas filogeneticamente dos vegetais
superiores, apresentam pectinas na parede celular (Domozych et al 2007). Por outro lado, outras
microalgas verdes apresentam parede celular e polissacarídeos extracelulares com características
semelhantes às pectinas, como as “pherophorins”, glicoproteínas que formam o envelope celular
típico das Ordens Volvocales e Chlamydomonadales (Morita et al 1999). A atividade
imunoestimuladora de Chlorella (Chlorococcales) é diretamente relacionada à presença de
polissacarídeos ricos em arabinanos, ramnanos e galactanos neste gênero (Kralovec et al 2007,
Suárez et al 2008), evidenciando as algas verdes como fontes naturais, além de facilmente
cultiváveis, de polímeros bioativos ricos em arabinose, galactose e ácido galacturônico. Em outro
exemplo, polissacarídeos são também responsáveis pela atividade imunoestimulatória de
RespondinTM, um imunoestimulante desenvolvido pela Ocean Nutrition Canada Ltd a partir de
extratos celulares de C. pyrenoidosa, dos quais Suárez et al (2005) isolaram um arabinogalactano.
Um estudo realizado com extratos brutos celulares e extracelulares de C. pumilioniformis
indicou efeito analgésico e anti-inflamatório bastante significativo em ambos os extratos com
inibição da resposta à dor superior às obtidas para morfina e efeito anti-inflamatório semelhante ao
diclofenaco (Andrade 2010). Análises da composição de polissacarídeos celulares e extracelulares
produzidos por C. pumilioniformis já foram realizadas em trabalhos de conclusão de curso e
dissertações de mestrado desenvolvidas em nosso laboratório (Maria et al 2011) e apontaram
polissacarídeos com potencial bioatividade ricos em arabinose, galactose e ácido galacturônico,
porém realizados em culturas não axênicas e sem o fracionamento e a caracterização mais detalhada
dos mesmos.
O objetivo deste trabalho foi isolar e caracterizar a composição monossacarídica de
diferentes frações dos polissacarídeos extracelulares produzidos por culturas axênicas de C.
pumilioniformis, com vistas à identificação de frações potencialmente bioativas.
12
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Part 1: Isolation and biological assessment in vitro. Phytomedicine 14:57-64
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14
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Suárez ER (2008) First isolation and structural determination of cyclic b-(1→2)-glucans from an
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15
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Polymers 79:811-817
16
3) MANUSCRITO SUBMETIDO À REVISTA JOURNAL OF APLLIED PHYCOLOGY
Lucas da Silva Maria¹ • Danilo Giroldo²* Extracellular polysaccharides produced by Chlamydomonas cf. pumilioniformis (Chlamydophyceae): identification of potentially bioactive fractions. 1,2- Laboratório de Botânica Criptogâmica, Instituto de Ciências Biológicas, Universidade Federal do Rio Grande - FURG, Rio Grande, RS, 96203-900, Brasil. 1- [email protected] 2- [email protected], 021 53 84246125 * Corresponding Author
17
Abstract Microalgae are very diverse organisms which occupy several habitats and produce a wide range of
compounds with potential application, including polysaccharides. This study aimed to characterize the extracellular
polysaccharides produced by Chlamydomonas cf. pumilioniformis (Chlamydophyceae) in axenic batch cultures. It was
isolated from a small subtropical shallow lake and it has been maintained in the microalgal culture collection of the
Institute of Biological Sciences at the Federal University of Rio Grande, RS, Brazil. Polysaccharide fractions were
obtained by ion exchange (DEAE Sepharose "Fast Flow") and size exclusion chromatography (Sephacryl S400) and
they were characterized by high performance ion chromatography with pulsed amperometric detection (HPIC-PAD).
We isolated six fractions and only in one of them, rich in glucose, ribose and fructose, arabinose was not found. The
five other fractions were mainly composed of arabinose, galactose and glucose in different proportions. Considering all
fractions, we also found minor proportions of fucose, rhamnose, N-acetyl-galactosamine, N-acetyl-glucosamine,
mannose, xylose, galacturonic acid and glucuronic acid. The high content of arabinose and galactose make such
polysaccharides similar to pectins from higher plants, which have a well known biological effect e.g. anti-inflammatory,
immunostimulatory and vasodilator. Our results reinforce the significance of such polysaccharide composition to
previously verified biological activity in mice of C. pumilioniformis crude extract.
Key-words Volvocales, carbohydrates, chromatography, arabinose.
18
Introduction
Microalgae produce a large variety of biochemical compounds with potential biotechnology applications in the food,
cosmetic and pharmaceutical industries, as well as in energy production (Olaizola 2003). Among the phytoplankton
metabolites, polysaccharides are significant since they represent 40% to 90% of the organic compounds produced by
these organisms and exhibit a wide range of structures and compositions (Myklestad 1995).
The production of both intra and extracellular polysaccharides is a ubiquitous phytoplanktonic process
(Paulsen and Vieira, 1994). Most of intracellular polysaccharides play storage and structural roles. The first are usually
composed of homogeneous glucans, such as starch, chrysolaminarin and paramylon (Percival 1979, Bäumer et al.
2001), while the latter are also largely homogenous, especially the cell wall constituents such as xylans, mannans,
rhamnans (Carlberg and Percival, 1977, Hoek et al. 1995). On the other hand, extracellular polysaccharides (EPS) could
be homogeneous compounds, such as fucans (Giroldo and Vieira 2002), mannans (Vieira et al. 2006), and
arabinogalactans (Kiemle et al. 2007), or very heterogeneous compounds with five or more different monosaccharides
(Giroldo and Vieira 2005). EPS have been less studied than structural and storage polysaccharides with regard to their
composition and structure and their biological functions (Giroldo et al. 2007).
Microalgae are able to produce pectic polysaccharides with biological activity, which are characterized by the
presence of arabinose, galactose, rhamnose and uronic acids (Paulsen 2001, Gronhaug 2010). Both the neutral pectins,
such as arabinans and arabinogalactans, and the acid pectins, such as ramnogalacturans, are very frequent in medicinal
plants, being one of the main interests of pharmacognosy. Vascular plants such as Biophytum petersianum and Panax
ginseng have been used in immune system related treatments, e.g. joint paints, inflammation and malaria, and their
activity has been related to acid and neutral pectic polysaccharides (Inngjerdingen et al. 2008, Yu et al. 2010).
Several green algae show pectins in their cell wall composition, especially those closer to higher plants
(Domozych et al. 2007). For instance, Chlamydomonadales have pherophorins attached to their cell wall, which are
pectic glycoproteins of the extracellular matrix (Morita et al. 1999, Hallman 2006). Also the immunostimulatory
activity of Chlorella spp. (Chlorococcales) is related to the presence of pectic polysaccharides in this gender (Kralovec
et al. 2007, Suárez et al. 2008). Such characteristics make green algae a natural and easily cultivable source of bioactive
polymers rich in arabinose, galactose and galacturonic acid. Suárez et al. (2005) have isolated an arabinogalactan from
RespondinTM, an immunostimulant developed by Ocean Nutrition Canada Ltd from cellular material of Chlorella
pyrenoidosa.
A previous study performed with crude cellular and extracellular extracts of Chlamydomonas cf.
pumilioniformis has indicated strong antinociceptive and antiinflamatory effects in both extracts (Andrade 2010). Also
Maria et al. (2011) identified the production of polysaccharides rich in arabinose, galactose and galacturonic acid in
non-axenic C. pumilioniformis cultures, however without a more detailed characterization of such compounds. The aim
of this study was to isolate and characterize the different fractions of extracellular polysaccharides produced by axenic
cultures of C. pumilioniformis, for the identification of potentially bioactive ones.
Material and Methods
Organisms and culture conditions
Chlamydomonas cf. pumilioniformis L. Petérfi was isolated directly on the microscope from a subtropical shallow pond
(Polegar Pond, surface area of 10,000 m2) located in the Carreiros campus of the Federal University of Rio Grande, Rio
Grande, RS, Brazil. The cultures were grown in WC medium (Guillard and Lorenzen, 1972), under 100 µmol m2 s1
(photosynthetically active radiation), with a 10:14 h dark : light cycle and a temperature of 22°C ±1°C, in the
microalgal culture collection of the Institute of Biological Sciences at the Federal University of Rio Grande, RS, Brazil.
19
Axenic cultures of were obtained by washing the cultures with Dakin solution (Vieira, 1983), followed by several re-
isolations. Bacterial contamination tests were conducted regularly with WC medium plus glucose and peptone at the
concentration of 250 mg L-1 each.
Exopolysaccharides isolation and characterization
A 9 L C. pumilioniformis culture in the stationary growth phase (40 d, 6 x 106 cells ml-1) was centrifuged (15 min at
4000 rpm). The extracellular material was filtered with fiber glass filters 3 (Macherey-Nagels, Düren, Germany) and
concentrated to 250 mL in a rotary evaporator at 40 °C, before being dialyzed against distilled water in dialysis tubes
with a 12-14 kD molecular weight cut-off. The dialyzed material was liofilized and stored at -4°C.
The freeze-dried material was further separated by anion exchange and size exclusion column chromatography
and the monosaccharide composition of the fractions was determined by high performance ion chromatography with
pulse amperometric detection (HPIC-PAD) as described below. The anion exchange column chromatography was
performed using the batch separation method, without gradient, under the following conditions: gel = Sepharose DEAE
fast flow (Pharmacia, Peapack, NJ, USA), eluent I = distilled water, eluent II = 0.5M NaCl, eluent III = 1.0M NaCl, and
eluent IV = 2.0M NaCl, running at the ambient temperature. Sodium azide (1 g L-1) was used to avoid bacterial
contamination. The column was first regenerated with 2.0M NaCl and washed with distilled water. The sample was
applied and eluted with distilled water to obtain the neutral fraction. Elution with 0.5M NaCl then yielded the weak acid
fraction, and finally elution with 1.0M NaCl was used to obtain the strong acid fraction. The size exclusion
chromatography was performed using the gel Sephacryl S-400 (Pharmacia, Peapack, NJ, USA) and butanol 2% as
eluent, with a load volume of 5% of the total volume of the column. The column was calibrated using dextrans of 5x105
and 2x106 D. For both chromatographic techniques, the flow rate was of 0.3 mL min -1 and carbohydrates were detected
in the 2-mL fractions by the phenol-sulfuric method (Dubois et al. 1956). The fractions with significant amount of
carbohydrates were pooled, dialyzed against distilled water, and freeze dried. After hydrolysis (Gremm and Kaplan
1997) the HPIC-PAD analyses were performed on a Dionex® ICS3000 device (Sunnyvale, CA, EUA) equipped with
PA-1 (Dionex) a anion-exchange analytical column (2 x 250 mm) with a corresponding guard-column (2 x 50 mm). The
eluent used for the separation of the monosaccharides was NaOH 18 mM, with 200 mM NaOH for column
recuperation, at a flow rate of 0.25 mL min -1 (Gremm and Kaplan 1997).
Results
Figure 1 shows two fractions of the EPS released by C. pumilioniformis obtained by anion exchange chromatography
using DEAE Sepharose gel. The neutral fraction, eluted by distilled water, and the acid fraction, eluted by 0.5M NaCl,
corresponded to 54.1% and 45.8% of the originally injected material. Figure 3 shows the molecular weight distribution
of the EPS of C. pumilioniformis obtained by size exclusion chromatography using Sephacryl S-400 gel. The arrow
shows a peak of dextran 5 x 105 D (Ve.Vt-1 = 0.19). Three neutral fractions were obtained, A (Ve.Vt-1 = 0.16 to 0.6), B
(0.62 to 0.69) and C (0.71 to 0.92), besides three acid fractions, D (0.45 to 0.62), E (0.63 to 0.77) and F (0.78 to 0.92).
Table 1 shows the recuperated fractions and percentages in relation to the original 40 mg of polysaccharide.
Fraction A was the most representative, corresponding to 38.7% of the total polysaccharide, followed in by fractions D
(21%), E (17%), C (8.7%), F (7.9%) and B (6.7%). The three neutral fractions of the polysaccharide are essentially
composed of arabinose, galactose and glucose (Figure 3).
Fraction A was mainly composed by arabinose (53.8%) and galactose (21.2%), with small amounts of
rhamnose, glucose, mannose and xylose. Fraction B also presented high percentages of arabinose (30%) and galactose
(13%), although it was mainly composed by glucose (45.5%). Fraction C presented the same profile as fraction B, but
20
with different quantities of the three main monosaccharides (arabinose – 31%, galactose – 18%, glucose – 35%). No
uronic acids were detected on these neutral fractions and we found three unidentified monosaccharides, with retention
times between fucose and rhamnose. Fraction D is a very heterogeneous polysaccharide, rich in arabinose (27%) and
galactose (13%), also containing significant amounts of mannose, xylose, glucose and rhamnose, as well as minor
amounts of fucose, N-acetyl-glucosamine and galacturonic and glucuronic acids. Fraction E has a more homogeneous
profile, composed of arabinose and galactose in higher amounts (25% each), followed by glucose and xylose (12.3%)
and minor amounts of fucose, rhamnose, mannose and uronic acids (never exceeding 7%). Also several unidentified
acids similar to the uronic acids were observed. Fraction F is very homogeneous, with a composition dominated by
glucose (56.9%) and ribose (18.9%), containing small amounts of arabinose, galactose and fructose and no uronic acids.
Discussion
This study confirms and expands the knowledge on the EPS of C. pumilioniformis, which presented five
polysaccharidic fractions rich in arabinose and galactose, with minor proportions of rhamnose, fucose, mannose and
xylose, as well as uronic acids. Polysaccharides such as the produced by C. pumilioniformes are similar to pectins
produced by vascular plants, which include arabinogalactans tipe I and tipe II, differentiated by the location of the
linkage or ramification carbon. In a study with leaf polysaccharides of the african Opilia celtidifolia, commonly used
for wound healing, Gronhaug et al. (2010) isolated eight acid fractions of pectic polysaccharides with
immunostimulatory activity. Such results indicate that the biological effect of the extracellular medium of C.
pumilioniformis in mice, demonstrated by Andrade (2010), may be related to the pectic composition of the
polysaccharides excreted by the algae. The results of Gronhaug et al. (2010) are not only similar to ours in the presence
of arabinogalactans and ramnogalacturans, but also in the fact that these polysaccharides present various
arabinose:galactose:rhamnose ratios, which shows the variety of pectic polysaccharides that can be produced by both
vascular plants and algae.
Although Paulsen and Barsett (2005) indicate that most bioactive pectic polysaccharides are charged due to the
presence of uronic acids, Yamada e Kiyohara (1999) isolated from Bupleurum falcatum a neutral pectic polysaccharide
with immunostimulatory activity. Those results show that the neutral fractions we found in C. pumilioniformis can be
promising compounds to pharmacological prospection.
The acid polysaccharide fraction D isolated in this study showed a very heterogeneous composition, which
indicates the efficiency of the hydrolytic extraction of its monosaccharides, as well as a complexity-based potential for
bioactivity. On the other hand, the larger proportion of this fraction (38.9%), as well as its wide spread peak, indicate
the need for further purification of this polysaccharide, using different chromatographic conditions. The acidic
characteristic of this fraction is confirmed by the presence of galacturonic and glucuronic acids in significant amounts
(9% and 10%, respectively). Uronic acids have negative charge and a well known potential for metal complexation
(Gouvêa et al. 2005), which may be another biotechnological potential of the polysaccharides of C. pumilioniformis.
Fraction E showed smaller amounts of galacturonic and glucuronic acids than fraction D, nevertheless it showed peaks
adjacent to these acids, whose identification may be further performed by comparison with other acids commonly found
in microalgae, such as the mannuronic acid.
Fraction F was highly homogeneous, being composed in more than 75% of glucose and ribose, and may be
originated from the intracellular material of the algae. A previous study (Maria 2011) showed that glucose is the most
abundant monosaccharide in the interior of the cell of three algae, including C. pumilioniformis. Furthermore, previous
data suggests that the presence of ribose is an indication of cellular decomposition (Veuger et al 2012). Although this
fraction was obtained by elution with NaCl 0.5M, which characterizes it as an acid fraction, no acid sugar had been
21
found nd probably sch acid feature was given by another radical, such as sulfate.
In conclusion, axenic cultures of C. pumilioniformis produced five different polysaccharide fractions with
pectic composition, which indicate them as a source of bioactive compounds with pharmacological interest.
Furthermore, the microalga presents a uronic acid rich polysaccharide with the potential biotechnological application of
metal complexation.
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(Volvocales). Planta 208:365-372
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Total Environ 165:155-164
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Biomol Eng 20:459-466
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characterization and use. Springer-Verlag, Berlin, 186:69-101
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Spondylosium panduriforme. J Phycol 30:638-641
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arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa. Carbohyd Res 340:1489–1498
Suárez ER, Bugden SM et al. (2008) First isolation and structural determination of cyclic b-(1→2)-glucans from an
alga, Chlorella pyrenoidosa. Carbohyd Res 343:2623–2633
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of Aulacoseira granulata (Bacillariophyceae). Acta Limnol Bras 18:1-7
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degradation products as determined by a sensitive high-performed ion chromatography method. Appl Environ Microb
23
57:3135- 3143
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Comprehensive Glycoscience–From Chemistry to Systems Biology. Oxford, Elsevier
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Table 1. Distribution of the extracellular polysaccharide fractions produced by C. pumilioniformis isolated by anion
exchange chromatography (Sepharose “Fast Flow” Pharmacia®) and size exclusion chromatography (Sephacryl S400
Pharmacia®).
Fraction Mass (mg) %
A 15,48 38,7
B 2,69 6,7
C 3,49 8,7
D 8,38 21
E 6,79 17
F 3,16 7,9
Total 40 mg 100%
24
0,0 0,2 0,4 0,6 0,8 1,0
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
0,0 0,2 0,4 0,6 0,8 1,0
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
Ve.Vt-1
H20
0,5 M
Ab
so
rban
ce
Figure 1. DEAE Sepharose (Pharmacia®) gel column ion exchange chromatography of the polysaccharides released by
C. pumilioniformis. The eluents used were distilled water and 0,5 M NaCl. Ve.Vt-1 = eluted volume divided by total
column volume.
25
0,0 0,2 0,4 0,6 0,8 1,0
0,00
0,01
0,02
0,03
0,04
0,0 0,2 0,4 0,6 0,8 1,0
0,00
0,01
0,02
0,03
0,04
a b
ba
Ve.Vt-1
F
DE
BC
A
0,5 M
H2O
A
bso
rban
ce
Figure 2.
Sephacryl S400 (Pharmacia®) gel size exclusion chromatography of the acid (0.5 M) and neutral (H2O) polysaccharides
released by C. pumilioniformis The arrow (a) indicates a peak of dextran 2x106 D. The arrow (b) indicates a peak of
dextran 5x105 D. Ve.Vt-1 = eluted volume divided by total column volume.
26
Figure 3. High performance ion chromatography with pulse amperometric detection (HPIC-PAD) of the neutral
polysaccharide fractions A, B and C produced by C. pumilioniformis.
27
Figura 4. High performance ion chromatography with pulse amperometric detection (HPIC-PAD) of the acid
polysaccharide fractions D, E and F produced by C. pumilioniformis.
28
0
20
40
60
C
B
A
0
20
40
60
fuc
rha
Ng
al
ara
Ng
lug
al
glu
man
xyl
fru
rib
ag
al
ag
lu
0
20
40
60
0
20
40
60
F
E
D
0
20
40
60
fuc
rha
Ng
al
ara
Ng
lug
al
glu
man
xyl
fru
rib
ag
al
ag
lu
0
20
40
60
Figure 5. Monosaccharide percentual composition of the neutral (A, B and C) and acid (D, E and F) polysaccharide
fractions of C. pumilioniformis. fuc = Fucose, rha = Rhamnose, Nagal = N-Acetil-galactosamine, ara = Arabinose,
Naglu = N-Acetil-glicosamine, gal = Galactose, glu = Glicose, man = Mannose, xyl = Xilose, fru = Fructose, rib =
Ribose, agal = Galacturonic Acid, aglu = Glucuronic Acid.
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4) INSTRUÇÕES DA REVISTA JOURNAL OF APPLIED PHYCOLOGY
Manuscript Submission Submission of a manuscript implies: that the work described has not been published before; that it is not under consideration for publication anywhere else; that its publication has been approved by all co-authors, if any, as well as by the responsible authorities – tacitly or explicitly – at the institute where the work has been carried out. The publisher will not be held legally responsible should there be any claims for compensation.
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Abbreviations Abbreviations should be defined at first mention and used consistently thereafter.
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• Negotiation research spans many disciplines (Thompson 1990). • This result was later contradicted by Becker and Seligman (1996). • This effect has been widely studied (Abbott 1991; Barakat et al. 1995; Kelso and Smith
1998; Medvec et al. 1999).
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• Journal article Gamelin FX, Baquet G, Berthoin S, Thevenet D, Nourry C, Nottin S, Bosquet L (2009) Effect of high intensity intermittent training on heart rate variability in prepubescent children. Eur J Appl Physiol 105:731-738. doi: 10.1007/s00421-008-0955-8 Ideally, the names of all authors should be provided, but the usage of “et al” in long author lists will also be accepted: Smith J, Jones M Jr, Houghton L et al (1999) Future of health insurance. N Engl J Med 965:325–329
• Article by DOI • Slifka MK, Whitton JL (2000) Clinical implications of dysregulated cytokine production. J
Mol Med. doi:10.1007/s001090000086 • Book • South J, Blass B (2001) The future of modern genomics. Blackwell, London • Book chapter • Brown B, Aaron M (2001) The politics of nature. In: Smith J (ed) The rise of modern
genomics, 3rd edn. Wiley, New York, pp 230-257 • Online document • Cartwright J (2007) Big stars have weather too. IOP Publishing PhysicsWeb.
http://physicsweb.org/articles/news/11/6/16/1. Accessed 26 June 2007 • Dissertation
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• Trent JW (1975) Experimental acute renal failure. Dissertation, University of California • Always use the standard abbreviation of a journal’s name according to the ISSN List of Title
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Color Art • Color art is free of charge for online publication. • If black and white will be shown in the print version, make sure that the main information
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33
Accessibility In order to give people of all abilities and disabilities access to the content of your figures, please make sure that
• All figures have descriptive captions (blind users could then use a text-to-speech software or a text-to-Braille hardware)
• Patterns are used instead of or in addition to colors for conveying information (color-blind users would then be able to distinguish the visual elements)
• Any figure lettering has a contrast ratio of at least 4.5:1 Fonte: Journal of Applied Phycology – Instructions for Authors.
http://www.springer.com/life+sciences/plant+sciences/journal/10811 Último Acesso: 8-7-2012.
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5) ANEXO: MEIO WC
pH 7,0 ± 0,5 (Guillard & Lorenzen, 1972) 1. CaCl2 36,8 g/L 1 mL/L 2. MgSO4 37 g/L 1 mL/L 3. NaHCO3 12,6 g/L 1 mL/L 4. NaSiO3 28,4 g/L 1 mL/L 5. K2HPO4 8,7 g/L 1 mL/L 6. NaNO3 85 g/L 1 mL/L 7. Micronutrientes 1 mL/L
Na2EDTA 4,36 g/L FeCl3 3,15 g/L CuSO4 0,01 g/L ZnSO4 0,022 g/L CoCl2 0,01 g/L MnCl2 0,018 g/L Na2MoO4 0,0006 g/L H3BO3 0,1 g/L
8. Vitaminas 1 mL/L
Tiamina 0,1 g/L Biotina 0,0005 g/L B12 0,0005 g/L
No “WC P+G”, adicionam-se 250 mg L-1 de peptona e 250 mg L-1 de glicose.