ABSTRACT
Abstract The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)'2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)'2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.
ABSTRACT
Introdução: A procura por novas alternativas terapêuticas, como as que utilizam as plantas medicinais, tem despertado grande interesse da comunidade científica na busca por tratamentos mais eficientes para as doenças, incluindo o câncer. Terminalia fagifolia Mart. é uma planta medicinal encontrada no Cerrado brasileiro, usada popularmente no tratamento de aftas e tumores. Objetivos: Avaliar a atividade citotóxica dos extratos etanólicos da casca e das folhas da Terminalia fagifolia em linhagens celulares NIH 3T3 e L929 e tumorais PC3 e B16F10. Métodos: Foi realizada a metodologia de determinação da viabilidade celular em ensaio com monocamada de células utilizando o ensaio MTS. As linhagens NIH 3T3, L929, PC3 e B16F10 foram expostas por 24 horas a diferentes concentrações dos extratos etanólicos da casca e folhas da Terminalia fagifolia. Resultados: Os resultados adquiridos mostraram que os extratos apresentaram viabilidade celular, sendo considerada de moderada a alta, para as células normais NIH 3T3 e L929 e citotoxicidade severa para as células tumorais PC3 e B16F10. Dessa forma, torna-se necessária a continuidade dos estudos com essa planta, pois os extratos da casca e das folhas apresentaram atividades antitumorais muito promissoras. Conclusões: Os extratos da casca e das folhas demonstraram viabilidade celular ≥ 50% nas linhagens celulares normais NIH 3T3 e L929 e demonstraram atividade citotóxica para as linhagens tumorais PC3 e B16F10, apresentando redução da viabilidade celular em torno de 60% e 70%, respectivamente. (AU)
Introduction: The search for new therapeutic alternatives, as the ones that use medicinal plants, has awaken a huge interest from the scientific community in seeking through more efficient treatments for diseases, including cancer. Terminalia fagifolia Mart. is a medicinal plant found in Brazilian "Cerrado", popularly used in aphthas and tumor treatment. Objectives: To evaluate the cytotoxic activity of ethanolic extracts of the bark and leaves of the Terminalia fagifolia in cell lines NIH 3T3 and L929 and tumor cells PC3 and B16F10. Methods: The determination methodology in cellular viability was held in an assay with cells monolayer's using the MTS assay. The NIH 3T3, L929, PC3 and B16F10 lines was exposed for 24 hours in differents ethanolic extracts concentrations. Results: The acquired results showed that the extracts had cellular variability is considered moderate to high for the normal cells NIH 3T3 and L929 and severe cytotoxicity to tumor cells PC3 and B16F10. This way, it is necessary to continue studying this plant, since both the bark and leaves extracts have great antitumor activity. Conclusion: The bark and leaves extracts showed cellular variability ≥ 50 % in normal cell lines NIH 3T3 and L929 and demonstrated cytotoxic activity for tumor cells PC3 and B16F10, presenting reduction of cell variability around 60% and 80%, respectively. (AU)