Laboratory of Biochemistry and Function of Plant Proteins, Department of Botany, Federal University of the State of Rio de Janeiro, Av. Pasteur, 458, Urca, Brazil.
Laboratory of Genotoxicity, Department of Genetics and Molecular Biology, Federal University of the State of Rio de Janeiro, R. Frei Caneca, 94 - Centro, Brazil.
J Ethnopharmacol. 2022 Mar 1;285:114903. doi: 10.1016/j.jep.2021.114903. Epub 2021 Dec 8.
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.
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.
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.
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.
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.
大戟属植物是一种热带和亚热带植物，也被俗称为“avelós”，民间医学用其治疗许多疾病，如风湿、哮喘、牙痛和癌症。研究表明，这种植物物种中含有的天然化合物可能与这些功能有关。然而，人们对其潜在毒性知之甚少。
几种蛋白质发挥着生物功能，特别是蛋白酶，在包括植物、动物和微生物在内的许多生物机制中起着至关重要的作用。然而，当它们调控不良时，可能会产生一些后果，如某些物质无法产生，甚至细胞异常增殖，从而导致肿瘤。另一方面，蛋白酶抑制剂通过降低蛋白酶的活性来调节这些酶，从而防止其功能失调。这项工作的目的是评估大戟属植物茎蛋白提取物的毒性，并纯化可能与植物生物医学功能相关的蛋白酶抑制剂。
使用艾姆斯试验研究了从“avelós”茎中提取的蛋白质提取物的细胞毒性和致突变特性。还使用凝胶过滤层析技术对蛋白质提取物进行了蛋白酶抑制剂的纯化过程，并对纯化的蛋白质进行了生化特性分析。
分离出一种名为 tirustatin 的蛋白酶抑制剂，其在 Biogel P100 上的得率为 1.84 倍。分离出的蛋白质的计算分子量为 25.97 kDa。抑制剂在 pH 3-10 之间稳定，在 pH 6 时活性显著。即使在高温（100°C）下也表现出热稳定性，与室温下的抑制剂活性相比，抑制活性增加了 1.14 倍。在碱性 pH 值下孵育长达 60 分钟，对 tirustatin 的木瓜蛋白酶抑制活性的降低很少（0.25 倍）。木瓜蛋白酶-tirustatin 相互作用的化学计量比为 1.5:1，28.8 pM 的抑制剂使 50%的酶失活。由于对木瓜蛋白酶的酶的平衡解离常数为 8.74 x 10-8M，可以评估分离出的蛋白质为非竞争性抑制剂。此外，即使在细菌模型中使用最大浓度（20μg/mL），大戟属植物的蛋白质提取物也没有表现出细胞毒性和致突变特征。
本工作提供的数据加强了利用大戟属植物中产生的蛋白质作为药理学和生物技术制剂的潜在用途的观点，这些制剂可用于开发有效的药物。
