Atividade antitumoral, isolamento e identificação dos principios ativos da Gaylussacia brasiliensis (Spreng.) Meisn. (Ericaceae)
TESE
Português
(Broch.)
T/UNICAMP An88a
Campinas, SP : [s.n.], 2004.
144p. : il.
Orientador: Paulo Eduardo Pizão
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Resumo: Gaylussacia brasiliensis (Spreng.) Meisn. (Ericaceae), popularmente conhecida corno "camarinha", é usada na medicina popular brasileira no tratamento de processos inflamatórios. Os objetivos deste trabalho foram: (1) avaliar a atividade antitumoral de extratos brutos obtidos desta espécie...
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Resumo: Gaylussacia brasiliensis (Spreng.) Meisn. (Ericaceae), popularmente conhecida corno "camarinha", é usada na medicina popular brasileira no tratamento de processos inflamatórios. Os objetivos deste trabalho foram: (1) avaliar a atividade antitumoral de extratos brutos obtidos desta espécie vegetal em modelo "in vitro" de cultura de células tumorais humanas; (2) determinar as frações ativas farmacologicamente nos modelos de cultura de células tumorais humanas e isolar e caracterizar quimicamente o(s) princípio(s) ativo(s) responsável(eis) por esta atividade; (3) avaliar a correlação entre o mecanismo responsável pela atividade antitumoral do princípio ativo isolado de Gaylussacia brasiliensis (Spreng.) Meisn. e a inibição da enzima cic1ooxigenaseem modelos "in vitro" e "in vivo" e, (4) avaliar o mecanismo responsável pela atividade antitumoral do princípio ativo isolados de Gaylussacia brasiliensis Meiss. em modelo "in vitro". A atividade antiproliferativa do extrato bruto diclorometânico (EBD) foi avaliada no ensaio da sulforrodamina B em quatro concentrações (250, 25, 2.5, 0.25 Ug/mL), após 48 horas de incubação. EBD inibiu o crescimento de todas as linhagens celulares, de forma concentração dependente, com melhor atividade citocida sobre a linhagem celular NCI-ADR. Então, EBD foi pré-purificado em coluna cromatográfica seca em silica-gel fornecendo 5 frações. Dentre estas, a fração C (PC) foi a mais ativa, inibindo todas as células na concentração de 25Ug/mL. FC foi fracionada em silica-gel com uma mistura de hexano-acetato de etila, originando o composto puro MAA-266. Este composto foi identificado com base em experimentos 2D-NMR como um triterpeno (acido 2 ß, 3 ß dihidroxi-urs-12-ene-28-oic). MAA-266 apresentou atividade antirproliferativa concentração-dependente e seletividade para as linhagens celulares 786-0 e MCF-7; o menor valor de IC5o foi de 6 Ug/mL para a linhagem celular NCI-ADR. Vários trabalhos têm demonstrado a participação do ácido acetilsalicílico na redução dos riscos de câncer de cólon intestinal e estudos in vitro e in vivo tentam avaliar o mecanismo de ação antiproliferativo dos inibidores específicos da COX-2, provavelmente relacionado à indução da apoptose celular e o bloqueio da produção de PGE2.A avaliação de uma ação inibitória de EBD sobre a síntese da PGE2 em mucosa gástrica de ratos e de MAA-266 sobre a síntese da PGE2 em célula tumoral humana HT-29, sugeriu que o mecanismo de ação antiproliferativo poderia envolver a biossíntese dos eicosanóides. Por outro lado, foram analisadas outras hipóteses a fim de evidenciarmos qual o mecanismo de ação responsável pela atividade antiproliferativa do composto MAA-266 obtido de Gaylussacia brasiliensis (Spreng.) Meisn., como possíveis ações no DNA e na indução da apoptose. Assim, investigamos se a atividade antiproliferativa de MAA- 266 era dependente do tempo de exposição da célula ao mesmo, inferindo que a citotoxicidade de um fánnaco por interferência com o metabolismo dos ácidos nuc1éicos fosse um processo lento. Quando a célula tumoral humana OVCAR-03 foi exposta, em diferentes períodos de tempo, ao 3 composto MAA-266, não foi demonstrado um incremento do efeito antiproliferativo dependente do tempo de exposição, o que pode sugerir que MAA-266 não deva interferir em rotas metabólicas das bases púricas e pirimidínicas, funcionando como um antimetabólito. Além disso, a estrutura química do composto MAA-266 não apresenta similaridade com os metabólitos endógenos. Para investigar a hipótese de que o mecanismo de ação antiproliferativo do composto MAA-266 envolvesse a intercalação nos ácidos nuc1éicos, realizados o ensaio denominado "Teste do DNA-methylgreen". Os resultados obtidos demonstraram que o composto MAA-266 foi inativo, enquanto que a doxorrubicina foi capaz de reduzir a absorbância em 48%, sugerindo que o mecanismo de ação antiproliferativo de MAA-266 não deva envolver a intercalação do DNA. Finalmente, o efeito pró-apoptótico de MAA-266 foi avaliado através da análise morfológica de células 4a linhagem OYCAR-03, em microscopia ótica e eletrônica e varredura, expostas a diferentes concentrações deste composto (250, 50, 25 e 2,5 Ug/mL), por 48 horas. A análise microscópica ótica revelou fragmentação nuclear, vacuolização citoplasmática, muItinuc1eação e células gigantes. Já na microscopia eletrônica de varredura foi observado formação de vesículas na membrana celular com redução do volume da mesma, características estas associadas ao perfil apoptótico. A correlação entre os achados morfológicos e o mecanismo antiproliferativo está atualmente sendo investigado através do teste da anexina Y, assim como o isolamento de outros compostos ativos presentes nesta espécie vegetal
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Abstract: Gaylussacia brasiliensis (Spreng.) Meisn.(Ericaceae), popularly known as "camarinha", is used in Brazilian folk medicine for treatment of several inflammatory processes and as healing agent. The first scope of this work was to evaluate the antiproliferative activity of the crude...
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Abstract: Gaylussacia brasiliensis (Spreng.) Meisn.(Ericaceae), popularly known as "camarinha", is used in Brazilian folk medicine for treatment of several inflammatory processes and as healing agent. The first scope of this work was to evaluate the antiproliferative activity of the crude dichIoromethanic extract (CHD) obtained from G. brasiliensis and to identify the active compound(s) responsible for this activity. CHD was evaluated on sulforhodamine B
antiproliferative assay in four-Ievel concentration (250, 25; 2.5; 0.25 Ug/mL) for 48 hours incubation. CHD showed a concentration-dependent inhibition on alI cell lines, but had better cytocidal activity for NCI-ADR cell line. Therefore, CHD was submitted to column chromatography on silica-gel providing tractions that revealed activity on the antiproliferative assay employed. CHD was pre-purified by dry column chromatography over silica-gel affording tive fractions. Among these, fraction C (PC) was the most active inhibiting alI cells line at 25Ug/mL. FC was further fractionated over silica-gel with hexane-ethyl acetate mixtures affording pure Compound 1. This compound was identitied on basis of 2D-NMR experiments. Compound 1 showed concentration-dependent activity and selectivity for 786-0 and MCF-7 cancer celllines; the lowest lCso was for NCl-ADR line with 6 J.tglmL.Based on experimental evidence that inhibition of COX 2 causes tumor regression, the second scope of this work was to evaluate the correlationship between CHD and Compound 1's antiproliferative activityand COX inhibition. The CHD in vivo anti-inflammatory activity was analyzed through the antiedematogenic model induced by carrageenin in rats. CHD (1000 mg/kg, po) inhibited paw edema and its maximum inhibitory effect (44%) was obtained afier 5h of the carrageenin injection. Indomethacin (10 mg/kg, po) started to reduce the paw edema on the second hour afier the edema induction and a maximum inhibitory effect (64%) was obtained afier 4h of the injection. The systemic effect of CHD (500 mg/kg, po) over the synthesis of PGE2 in the gastric mucosa of rats, with or without previous indomethacin (5 mg!kg, sc) treatment, was measured. CHD inhibited 50% of its PGE2 synthesis, which was potentiated (82%) of indomethacin previous administration. To evaluate the same pharmacological activity of Compound 1 (250Ug/mL), the amount of PGE2 in humancancer celllines (HT-29 - colon) was measured, for 48 h. Both Compound 1 and indomethacin reduced the synthesis of PGE2 in vitro, at 64 and 57%, respectively. Overall, experimental data suggest that the Compound 1 antitumor mechanism is related to COX inhibition. The third scope of this work was to evaluate the antiproliferative activity mechanism of Compound 1 (2 ß, 3 ß dihydroxy-urs-12-ene-28-oic acid) isolated from G. brasiliensis (Spreng.) Meisn. (Ericaceae), employing the sulforhodamine B antiproliferative assay using OVCAR-03 (human ovary cancer) cell line. Its possible phannacological mechanisms of action were investigated inc1uding DNA syniliesis, cell division interference and its cap~city to induce in vitro cellular transformation. The DNA-MG bioassay revea1ed no signs of DNA intercalation. The time-independent (exposure irom 4 to 120 hours) antiproliferative activity over OYCAR-03 cellline exposed to this compound together with the morphological analysis revealed an apoptotic phenotype. These results suggest that the triterpene's both citotoxicity and ability to induce cellular transformation lead to apoptosis. The correlation between the morphological findings with changes in the optical properties of the cultures is currently being carried out using the annexin Y test as well as a bioassay guided isolation of other active components present in this planto Ver menos
antiproliferative assay in four-Ievel concentration (250, 25; 2.5; 0.25 Ug/mL) for 48 hours incubation. CHD showed a concentration-dependent inhibition on alI cell lines, but had better cytocidal activity for NCI-ADR cell line. Therefore, CHD was submitted to column chromatography on silica-gel providing tractions that revealed activity on the antiproliferative assay employed. CHD was pre-purified by dry column chromatography over silica-gel affording tive fractions. Among these, fraction C (PC) was the most active inhibiting alI cells line at 25Ug/mL. FC was further fractionated over silica-gel with hexane-ethyl acetate mixtures affording pure Compound 1. This compound was identitied on basis of 2D-NMR experiments. Compound 1 showed concentration-dependent activity and selectivity for 786-0 and MCF-7 cancer celllines; the lowest lCso was for NCl-ADR line with 6 J.tglmL.Based on experimental evidence that inhibition of COX 2 causes tumor regression, the second scope of this work was to evaluate the correlationship between CHD and Compound 1's antiproliferative activityand COX inhibition. The CHD in vivo anti-inflammatory activity was analyzed through the antiedematogenic model induced by carrageenin in rats. CHD (1000 mg/kg, po) inhibited paw edema and its maximum inhibitory effect (44%) was obtained afier 5h of the carrageenin injection. Indomethacin (10 mg/kg, po) started to reduce the paw edema on the second hour afier the edema induction and a maximum inhibitory effect (64%) was obtained afier 4h of the injection. The systemic effect of CHD (500 mg/kg, po) over the synthesis of PGE2 in the gastric mucosa of rats, with or without previous indomethacin (5 mg!kg, sc) treatment, was measured. CHD inhibited 50% of its PGE2 synthesis, which was potentiated (82%) of indomethacin previous administration. To evaluate the same pharmacological activity of Compound 1 (250Ug/mL), the amount of PGE2 in humancancer celllines (HT-29 - colon) was measured, for 48 h. Both Compound 1 and indomethacin reduced the synthesis of PGE2 in vitro, at 64 and 57%, respectively. Overall, experimental data suggest that the Compound 1 antitumor mechanism is related to COX inhibition. The third scope of this work was to evaluate the antiproliferative activity mechanism of Compound 1 (2 ß, 3 ß dihydroxy-urs-12-ene-28-oic acid) isolated from G. brasiliensis (Spreng.) Meisn. (Ericaceae), employing the sulforhodamine B antiproliferative assay using OVCAR-03 (human ovary cancer) cell line. Its possible phannacological mechanisms of action were investigated inc1uding DNA syniliesis, cell division interference and its cap~city to induce in vitro cellular transformation. The DNA-MG bioassay revea1ed no signs of DNA intercalation. The time-independent (exposure irom 4 to 120 hours) antiproliferative activity over OYCAR-03 cellline exposed to this compound together with the morphological analysis revealed an apoptotic phenotype. These results suggest that the triterpene's both citotoxicity and ability to induce cellular transformation lead to apoptosis. The correlation between the morphological findings with changes in the optical properties of the cultures is currently being carried out using the annexin Y test as well as a bioassay guided isolation of other active components present in this planto Ver menos
Atividade antitumoral, isolamento e identificação dos principios ativos da Gaylussacia brasiliensis (Spreng.) Meisn. (Ericaceae)
Atividade antitumoral, isolamento e identificação dos principios ativos da Gaylussacia brasiliensis (Spreng.) Meisn. (Ericaceae)
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