Abstract

BIOCHEMICAL PURIFICATION OF PROTEINS FROM BERTHOLLETIA EXCELSA SEEDS AND THEIR ANTILEISHMANIAL ACTION IN VITRO

Julia Miranda Fardin†, Laís Pessanha de Carvalho†, Viviane Veiga do Nascimento†, Edésio José Tenório de Melo, Valdirene M. Gomes, Olga Lima Tavares Machado, Flávia Camila Vieira da Silva and André de Oliveira Carvalho*

Abstract

Bertholletia excelsa is utilized as a folk medicine by the local population of the Amazon basin to treat several health problems. The aims of the study were to survey new protein extracts from B. excelsa seeds and test their antileishmanial actions in vitro. Proteins were extracted in phosphate buffer and submitted to DEAE-Sepharose. The antileishmanial activity of the obtained fractions was tested in growth inhibition assay against Leishmania amazonensis. The fraction that presented the strongest inhibitory activity was submitted to a new chromatographic step on DEAE-Sepharose with a new elution method. All new fractions were submitted to growth inhibition assay as before. The strongest fraction was further fractionated on a reverse-phase C18 column. The extraction and all chromatographic steps were monitored by gel electrophoresis. Protozoans were treated with the strongest fraction to determine the minimal inhibitory concentration, test for viability and assess the membrane permeabilization, oxidative state and mitochondria functionality. Selected fraction was characterized by Edman degradation amino acid sequencing and α-amylase inhibition was determined. Our results show DR2 fraction presented the strongest toxicity against L. amazonensis, causing 100% parasite elimination at 150 μg/mL. DR2 fraction toxic activity included membrane permeabilization, increased endogenous ROS production and mitochondria dysfunction. The main protein present in DR2 fraction is a member of the 2S albumin protein family. In conclusion, our results provide opportunities for drug design or development based on the proteins and peptides of B. excelsa that might be utilized to treat human leishmaniasis.

Keywords: Antileishmania, Chromatography, 2S albumin, Fluorescent microscopy, Minimal inhibitory concentration.