Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, São Paulo 09210-180, Brazil.
Department of Botany, Institute of Biosciences, University of São Paulo, São Paulo 05508-090, Brazil.
Phytomedicine. 2021 Dec;93:153748. doi: 10.1016/j.phymed.2021.153748. Epub 2021 Sep 11.
In the present work the bioactivity-guided fractionation of n-hexane extract from aerial parts of Baccharis sphenophylla (Asteraceae) against trypomastigote forms of Trypanosoma cruzi was performed.
To evaluate the antitrypanosomal potential of diterpenes ent‑kaurenoic (1), grandifloric (2). and 15β-tiglinoyloxy‑ent-kaurenoic (3) acids, isolated from n-hexane extract from aerial parts of B. sphenophylla, and elucidate their mechanism of action against T. cruzi. METHODS/STUDY DESIGN: n-Hexane and MeOH extracts from aerial parts of B. sphenophylla were prepared and caused, respectively, 100% and 50% of death of trypomastigote forms of T. cruzi. Based on these results, the n-hexane extract was subjected to bioactivity-guided fractionation procedures to afford three related ent‑kaurane diterpenoids (1-3). Based on spectrofluorometric assays and flow cytometry analysis, the mechanism of action of compounds 1 and 3 was investigated.
Compounds 1 and 3, isolated from n-hexane extract from aerial parts of B. sphenophylla, showed potent activity against parasites with EC values of 10.6 μM (SI > 18.8) and 2.4 μM (SI = 34.8), respectively. On the other hand, compound 2 was inactive against trypomastigotes. In mechanism of action studies using the fluorescent probe SYTOX Green, the plasma membrane permeability was unaltered after treatment with compounds 1 and 3, but compound 1 induced a depolarization of the plasma membrane electric potential (ΔΨp). No substantial alterations were observed in the mitochondria after treatment with compound 3, but a transient hyperpolarization of the mitochondrial membrane potential (ΔΨm) by compound 1. Despite the increased ATP levels induced by compounds 1 and 3, no alterations of ROS and Ca levels were registered. However, both compounds promoted a time-dependent alkalinization of the acidocalcisomes, probably contributing to an osmotic imbalance of the cell. In silico physicochemical studies of compounds 1-3 suggested that lipophilicity and molecular complexity may play an important role in the antitrypanosomal activity. Moreover, no pan-assay interference compounds (PAINS) alerts were detected for compounds 1-3.
Obtained data indicated that the isolated ent‑kaurane diterpenes from n-hexane extract from aerial parts of B. sphenophylla, especially compound 3, could be considered interesting prototypes for further modifications aiming the discovery of new hits against T. cruzi.
本工作对 Baccharis sphenophylla（菊科）地上部分的正己烷提取物进行了生物活性导向分离，以对抗克氏锥虫的无鞭毛体形式。
评估从 Baccharis sphenophylla 地上部分的正己烷提取物中分离得到的对映贝壳杉烯酸（1）、大波斯菊酸（2）和 15β- 亚油酸-对映贝壳杉烯酸（3）二萜的抗锥虫潜力，并阐明其对抗克氏锥虫的作用机制。
方法/研究设计：制备了 Baccharis sphenophylla 地上部分的正己烷和 MeOH 提取物，分别导致克氏锥虫的无鞭毛体形式 100%和 50%死亡。基于这些结果，对正己烷提取物进行了生物活性导向分离程序，得到了三种相关的对映贝壳杉烷二萜（1-3）。基于荧光分光光度法和流式细胞术分析，研究了化合物 1 和 3 的作用机制。
从 Baccharis sphenophylla 地上部分的正己烷提取物中分离得到的化合物 1 和 3 对寄生虫具有很强的活性，EC 值分别为 10.6 μM（SI > 18.8）和 2.4 μM（SI = 34.8）。另一方面，化合物 2 对无鞭毛体无活性。在使用荧光探针 SYTOX Green 的作用机制研究中，用化合物 1 和 3 处理后，质膜通透性没有改变，但化合物 1 诱导质膜去极化（ΔΨp）。用化合物 3 处理后，线粒体没有观察到明显的变化，但化合物 1 引起线粒体膜电位（ΔΨm）的短暂超极化。尽管化合物 1 和 3 诱导了 ATP 水平的升高，但未检测到 ROS 和 Ca 水平的改变。然而，这两种化合物都导致酸钙体的时间依赖性碱化，这可能导致细胞的渗透失衡。化合物 1-3 的计算物理化学研究表明，亲脂性和分子复杂性可能在抗锥虫活性中起重要作用。此外，化合物 1-3 未检测到泛分析干扰化合物（PAINS）警报。
获得的数据表明，从 Baccharis sphenophylla 地上部分的正己烷提取物中分离得到的对映贝壳杉烷二萜，特别是化合物 3，可能被认为是对抗克氏锥虫的新发现的有趣原型。
