Caracterização estrutural e atividade biológica de carboidratos de Phyllanthus niruri (quebra-pedra)

Abstract

Resumo: Este estudo foi desenvolvido com extratos de Phylanthus niruri, um fitoterapico de ampla distribuicao nas regioes tropicais e subtropicais do globo, popularmente conhecido como quebra-pedra. O trabalho traz uma abordagem quimica e biologica dos carboidratos que compoem a planta. O enfoque quimico trata do isolamento e caracterizacao de polissacarideos obtidos a partir de diferentes formas de extracao. Os extratos aquosos foram realizados nas formas de infusao, decoccao e extracao exaustiva a quente. A partir destes extratos foi isolada e caracterizada uma arabinogalactana pectica do tipo I, de estrutura complexa. Este polissacarideo apresentou cadeia principal constituida por unidades de b-Galp (1 - 4), parcialmente substituida por cadeias laterais contendo Rhap, GalpA, Araf, Galp e Glcp. A molecula apresentou terminais nao-redutores de Araf, Xylp, Rhap, Manp, Galp, e Glcp. Foram tambem realizadas extracoes alcalinas com KOH 2% que forneceram uma heteroxilana acida, tambem de estrutura complexa. A molecula apresentou cadeia principal constituida por unidades de b-Xylp (1 - 4), parcialmente substituida por cadeias laterais constituidas por unidades de Rhap, Araf e 4-O-Me-GlcpA. A molecula apresentou terminais nao-redutores de Araf, Xylp, Galp, Glcp e GlcpA. Com o intuito comparativo, foram tambem realizadas extracoes a partir dos orgaos vegetais separados: folhas, caules e raizes. Os extratos aquosos dos orgaos vegetais apresentaram grande similaridade, sugerindo a presenca de arabinogalactanas como principal componente. Ja os extratos alcalinos, obtidos com KOH 2% apresentaram algumas diferencas, especialmente quanto ao polissacarideo que compoe as folhas. Sugere-se que a amostra obtida das folhas seja constituida, majoritariamente, por uma arabinogalactana; ja os extratos obtidos dos caules e raizes sao constituidos por heteroxilanas, polissacarideo caracterizado ao se realizar extratos com a planta inteira. Foram ainda realizadas extracoes com NaOH 15% e os resultados obtidos apontam a presenca de xilanas substituidas e/ou xiloglucanas compondo o extrato das folhas, enquanto os dos caules e raizes sao constituidos por xilanas pouco substituidas. O enfoque biologico do trabalho foi desenvolvido com a arabinogalactana obtida a partir dos extratos aquosos realizados com a planta inteira. Os testes in vitro realizados em macrofagos peritoneais de camundongos apontaram que o polissacarideo e capaz de aumentar a producao de O2 - em 3 vezes, nao interferindo na producao de NO. Foi ainda observada uma atividade elicitora de celulas ao tratar os animais com o polissacarideo por 24 h, alcancando um aumento maximo de celulas no exsudato peritoneal de 2,3 vezes na dose de 100 mg.kg-¹. Por fim, o trabalho ainda avaliou o processo de digestao parcial da arabinogalactana, mimetizando condicoes acidas estomacais. Ao se submeter a arabinogalactana a hidrolises acidas a 37° C, por ate 3 h com suco gastrico e HCl foi observado que o polissacarideo sofre alteracoes quimicas decorrentes deste tratamento e que, ao contrario do esperado, o processo de digestao estomacal fragmenta a molecula de forma intensa. Fragmentos de alta e baixa massa molar da arabinogalactana parcialmente degradada foram tambem testados em macrofagos peritoneais de camundongos e foi observada uma ativacao maxima da producao de O2 - em 2 vezes para a fracao de baixa massa molar e 2,3 vezes para a fracao de alta massa molar, indicando uma relacao estrutura / atividade, na qual o tamanho, a conformacao e/ou a sequencia monossacaridica estao envolvidas.

This study was developed using extracts of Phyllanhtus niruri, known as stone-breaker (quebra-pedra), a medicinal herb widely distributed throughout tropical and subtropical regions. The work presents a chemical and biological focus on the carbohydrates present in the plant. The chemical approach deals with the isolation and characterization of polysaccharides obtained from different extraction procedures. Aqueous extractions were carried through in form of teas, as decoction and infusion, and exhaustively in hot water. From these extracts, a complex type I arabinogalactan was characterized. The pectic polymer presented a main chain constituted by a (1 - 4)-linked b-Galp, partially substituted by Rhap, GalpA, Araf, Galp e Glcp side chains, with nonreducing end units of Araf, Xylp, Rhap, Manp, Galp, and Glcp. Extractions using 2% aq. KOH were also carried out, affording an acidic heteroxylan with a complex structure. The polysaccharide had a (1 - 4)-linked b-Xylp mainchain, partially substituted by Rhap, Araf, and 4-O-methylglucuronic acid side chains, with nonreducing end units of Araf, Xylp, Galp, Glcp, and nonmethylated glucuronic acid. The next step was on the extraction and fractionation of polysaccharides from the different organs from the plant, aiming to compare the content of each organ. These results comprise samples obtained from leaves, steams, and roots. The aqueous extracts of the plant organs were very similar among themselves, suggesting the presence of an arabinogalactan as a main component. The alkaline fractions, obtained from 2% aq. KOH extractions, presented some differences, specially concerning the leaf extracts, which also contained an arabinogalactan as a major polysaccharide. On the other hand, the fractions obtained from the stems and roots were composed of a heteroxylan, similar to that of the whole plant. Extractions using 15% aq. NaOH were also carried out, and the extracts from the leaves presented substituted xylans and/or xyloglucans. The extracts from the stems and roots were very similar, in terms of the carbohydrate content, and afforded poorly substituted xylans. The biological approach of this study was carried out with the arabinogalactan, obtained from the aqueous extracts from the whole plant. The in vitro assays were conducted on the peritoneal macrophages of mice, and the polysaccharide was able to stimulate O2 - production in 3 fold, but did not interfere with the nitric oxide pathway. Cell eliciting activity was also observed in the polysaccharide-treated animals after 24 h treatment, achieving an increase of 2.3 times at a dose of 100 mg.kg-¹. A third approach of this work consisted of analyses of a partial digestion of the arabinogalactan, miming gastric conditions. The arabinogalactan was submitted to acid hydrolyses with HCl and gastric juice from human volunteers, at 37° C, for a maximum of 3 h. It was possible to observe a partial degradation of the polysaccharide to a much higher proportion than that expected. Fragments from the native polymer were also tested in the peritoneal macrophage of mice, and resulted in 2 and 2.3 fold activation of O2 - production for the low and high molar mass fragments, respectively. These results indicate a structure/activity relationship, in which the size, conformation and/or monosaccharide sequence may be involved.