Biochemical characterization and phytotoxic activity of protein extract from Euphorbia tirucalli L

Abstract

Ethnopharmacological relevance: Euphorbia tirucalli L., a tropical and subtropical plant, also known by the popular name avelós, has been used in folk medicine against many diseases as rheumatism, asthma, toothache, and cancer. Studies have shown that natural compounds contained in this plant species may be associated with these functions. However, little is known about its potential toxicity.

Aim of the study: Several proteins conduct biological functions, in particular, proteinases, play a crucial role in many mechanisms of living beings, including plants, animals and microorganisms. However, when poorly regulated, they can generate consequences, such as the non-production of certain substances, or even the abnormal multiplication of cells, which leads to tumors. On the other hand, by regulating these enzymes, proteinase inhibitors act by reducing the activity of proteinases, thus preventing their malfunction. The objective of this work was to evaluate the toxicity of the protein extract of E. tirucalli and to purify a protease inhibitor that may be associated with the biological medicinal functions of the plant.

Materials and methods: The cytotoxic and mutagenic properties of the protein extract produced from the stem of avelós was investigated using the Ames test. The protein extract was also submitted to a protease inhibitor purification process using the gel filtration chromatography technique and the purified protein was biochemically characterized.

Results: A protease inhibitor, called tirustatin, was isolated 1.84-fold by Biogel P100. The calculated molecular mass of the isolated protein is 25.97 kDa. The inhibitor was stable at pH 3-10, with pronounced activity at pH 6. Thermostability was observed even at elevated temperature (100 °C) with inhibitory activity increased by 1.14-fold compared to inhibitor activity at room temperature. Incubation at basic pH values for up to 60 min caused little reduction (0.25-fold) in the papain inhibitory activity of tirustatin. The stoichiometry of the papain-tirustatin interaction was 1.5: 1 and 28.8 pM of the inhibitor effected 50% inhibition. With an equilibrium dissociation constant of 8.74 x 10-8M for the papain enzyme, it is possible to evaluate the isolated protein as a non-competitive inhibitor. In addition, the protein extract of E. tirucalli even at the maximum concentration used (20 μg/mL), did not show a cytotoxic and mutagenic profile in a bacterial model.

Conclusion: The results presented in this work provide data that reinforce the idea of the potential use of proteins produced in E. tirucalli as pharmacological and biotechnological agents that can be exploited for the development of efficient drugs.

Keywords: Avelós; Cystatin; Cytotoxic activity; Protein extract.