Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, 09210-180, São Paulo, Brazil.
Centro de Parasitologia e Micologia, Instituto Adolfo Lutz, 01246-902, São Paulo, Brazil.
Phytomedicine. 2019 Feb 15;54:302-307. doi: 10.1016/j.phymed.2018.09.236. Epub 2018 Oct 9.
From a previous screening of Brazilian biodiversity for antitrypanosomal activity, the n-hexane extract from twigs of Nectandra oppositifolia (Lauraceae) demonstrated in vitro activity against Trypanosoma cruzi.
To perform the isolation and chemical characterization of bioactive compounds from n-hexane extract from twigs of N. oppositifolia and evaluate their therapeutical potential as well as to elucidate their mechanism of action against T. cruzi.
METHODS/STUDY DESIGN: Bioactivity-guided fractionation of the n-hexane extract from twigs of N. oppositifolia afforded three related butenolides: isolinderanolide D (1), isolinderanolide E (2) and secosubamolide A (3). These compounds were evaluated in vitro against T. cruzi (trypomastigote and amastigote forms) and against NCTC (L929) cells for mammalian cytotoxicity. Additionally, phenotypic analyzes of compounds-treated parasites were performed: alterations in the plasma membrane permeability, plasma membrane electric potential (ΔΨ), mitochondrial membrane potential (ΔΨ) and induction of ROS.
Compounds 1-3 were effective against T. cruzi, with IC values of 12.9, 29.9 and 12.5 µM for trypomastigotes and 25.3, 10.1 and 12.3 µM for intracellular amastigotes. Furthermore, it was observed alteration in the mitochondrial membrane potential (ΔΨ) of parasites treated with butenolides 1-3. These compounds caused no alteration to the parasite plasma membrane, and the deregulation of the mitochondria might be an early event to cell death. In addition, in silico studies showed that all butenolides were predicted to be non-mutagenic, non-carcinogenic, non hERG blockers, with acceptable human intestinal absorption, low inhibitory promiscuity with the main five CYP isoforms, and with high metabolic stability. Otherwise, tested butenolides showed unfavorable blood-brain barrier penetration (BBB+).
Our results demonstrated the anti-T. cruzi effects of compounds 1-3 isolated from N. oppositifolia and indicated that the lethal effect of these compounds in trypomastigotes of T. cruzi could be associated to the alteration in the mitochondrial membrane potential (ΔΨ).
从巴西生物多样性的抗锥虫活性筛选中，桃金娘科植物 Nectandra oppositifolia 的正己烷提取物显示出体外抗 Trypanosoma cruzi 的活性。
从 Nectandra oppositifolia 的小枝正己烷提取物中分离和化学表征具有生物活性的化合物，并评估其作为治疗药物的潜力，并阐明其对 T. cruzi 的作用机制。
方法/研究设计：对 Nectandra oppositifolia 的小枝正己烷提取物进行生物活性导向分离，得到三种相关的丁烯内酯：异缬草酸 D（1）、异缬草酸 E（2）和 secosubamolide A（3）。这些化合物在体外针对 T. cruzi（锥虫和阿米巴样体形式）和 NCTC（L929）细胞进行了哺乳动物细胞毒性评估。此外，还对化合物处理的寄生虫进行了表型分析：质膜通透性、质膜电势（ΔΨ）、线粒体膜电势（ΔΨ）和 ROS 的诱导变化。
化合物 1-3 对 T. cruzi 有效，对锥虫的 IC 值分别为 12.9、29.9 和 12.5 μM，对细胞内阿米巴样体的 IC 值分别为 25.3、10.1 和 12.3 μM。此外，还观察到丁烯内酯 1-3 处理的寄生虫中线粒体膜电势（ΔΨ）发生改变。这些化合物对寄生虫质膜没有改变，线粒体的去极化可能是细胞死亡的早期事件。此外，计算机模拟研究表明，所有丁烯内酯均被预测为非致突变、非致癌、非 hERG 阻断剂，具有可接受的人类肠道吸收性、对主要五种 CYP 同工酶的低抑制混杂性以及高代谢稳定性。另一方面，测试的丁烯内酯显示出不利于血脑屏障渗透（BBB+）的特性。
我们的结果表明，从 Nectandra oppositifolia 中分离得到的化合物 1-3 具有抗 T. cruzi 的作用，并表明这些化合物在 T. cruzi 的锥虫中的致死作用可能与线粒体膜电势（ΔΨ）的改变有关。
