Potencial anti-helmíntico e avaliação citotóxica in vitro de saponinas e flavonoides

Abstract

A identificação de novos compostos com potencial antiparasitário tem sido alvo de intensas investigações. Este trabalho teve como objetivo, avaliar o efeito in vitro de saponinas e flavonoides sobre nematoides gastrintestinais de caprinos e em cultivos de células de linhagem celular de rim de macaco verde africano (Vero). Foram testados saponinas (digitonina e escina) e suas respectivos aglicona (sapogeninas); sapogenina acetilada (acetato de hecogenina) e os flavonoides (catequina, hesperedina, isocordoína, mistura de isocordoína e cordoína, e dímero de chalcona). A atividade anti-helmíntica destes compostos foi avaliada através dos testes de inibição da eclosão de ovos (TEO) e motilidade larvar (TML). A ação citotóxica foi verificada empregando-se os ensaios de brometo de 3-(4,5-dimetiltiazol-2-il) - 2,5-difenitetrazólio (MTT) e iodeto de propídio (IP). Significativo percentual de inibição na eclosão de ovos (p<0.05), foi observado apenas nos tratamentos com a escina (99%) e a digitonina (45%). As CE50 e CE90 da escina foram respectivamente 0.67 e 0.79 mg/mL. No TML, os compostos que reduziram significativamente a motilidade de larvas (p<0.05) foram a digitonina em 95% (CE50 = 0.03 mg/mL e CE90 = 0.49 mg/mL) e o acetato de hecogenina em 75%. As agliconas da digitonina e escina apresentaram baixa atividade ovicida (3 e 8%) e larvicida (2 e 2%), respectivamente. Todos os flavonoides apresentaram baixo efeito ovicida (4 a 20%) e larvicida (10 a 26%). Apenas a escina e a digitonina, que apresentaram eficácia antihelmíntica igual ou acima de 95%, foram avaliados quanto à citotoxicidade; ambas demonstraram baixa toxicidade no teste de IP, sendo observado alto percentual de células integras de 94 e 96% respectivamente para a escina e digitonina, na maior concentração utilizada (0.03 mg/mL). No ensaio de MTT, estas saponinas foram citotóxicas, evidenciando 8% de células viáveis para a escina, na maior concentração (0.045 mg/mL) com CI50=0.20 mg/mL, e 6% de células viáveis para digitonina na maior concentração (0.083 mg/mL) com CI50 = 0.0074 mg/mL. Os resultados evidenciaram, que a escina e digitonina têm elevado efeito anti-helmíntico in vitro sobre nematoides gastrintestinais de caprinos, e que a presença das cadeias de açucares ligada aos núcleos dessas saponinas, aumentou esse efeito farmacológico.

The identification of new compounds with antiparasitic potential has been the target of intense investigations.This study aimed to evaluate the in vitro effect of saponins and flavonoids over gastrointestinal nematodes of goats and in cultured cells of the African green monkey kidney cell line (Vero).The tested compounds were saponins (digitonin and aescin) and their respective aglycone (sapogenin); the acetylated sapogenin (hecogenin acetate) and the flavonoids (catechin, hesperidin, isocordoin, a mixture of isocordoin and cordoin, Chalcone dimer). The anthelmintic activity of these compounds was evaluated through the inhibition of egg hatch assay (EHA) and larval motility assay (LMA). The cytotoxic action was verified through the employing of the 3-4,5- dimethylthiazol-2-yl,2,5 diphenyltetrazolium bromide (MTT) and propidium iodide (PI) assay. Significant reduction in the egg-hatching inhibition percentual (p<0.05) was observed only in the treatments with aescin (99%) and digitonin (45%). The EC50 and EC90 of aescin were 0.67 and 0.79 mg/mL, respectively. In the LMA test, the compounds which significantly reduced the larval motility (p<0.05) were digitonin with 95% (EC50= 0.03 mg/mL and EC90 = 0.49 mg/mL) and the hecogenin acetate with 75%. The sapogenins from digitonin and aescin did present low ovicidal (3 and 8%) and larvicidal (2 e 2%) activities, respectively. All the flavonoids showed a low ovicidal (4 to 20%) and larvicidal (10 to 26%) effect. Only the aescin and digitonin showed anthelmintic efficacy that was equal to or higher than 95% and, as such, were evaluated on their cytotoxicity; both showed low toxicity in the PI test, where a high percentage of whole cells 94% and 96%, for was observed aescin and digitonin, in the highest utilized concentration (0.03 mg/mL), respectively. In the MTT assay, these saponins showed cytotoxicity, with only 8% of the viable cells remaining after exposure to aescin in the highest concentration (0.045 mg/mL), with IC50=0.20 mg/mL; and 6% of the viable cells remaining after exposure to digitonin in the highest concentration (0.083 mg/mL) with IC50=0.0074 mg/mL.The results show that the aescin and digitonin have a pronounced in vitro anthelmintic effect over gastrointestinal nematodes of goats, and that the presence of sugar chains, bound to the core of these saponins, increased this pharmacological effect. Keyword: sapogenin;anthelmintic;aglycone;sugar chains;toxicity