Ent-kaurane diterpenes isolated from n-hexane extract of Baccharis sphenophylla by bioactivity-guided fractionation target the acidocalcisomes in Trypanosoma cruzi,Phytomedicine

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Ent-kaurane diterpenes isolated from n-hexane extract of Baccharis sphenophylla by bioactivity-guided fractionation target the acidocalcisomes in Trypanosoma cruzi
Phytomedicine ( IF 6.7 ) Pub Date : 2021-09-11 , DOI: 10.1016/j.phymed.2021.153748
Thais A da Costa-Silva ₁ , Matheus L Silva ₁ , Guilherme M Antar ₂ , Andre G Tempone ₃ , João Henrique G Lago ₁

Affiliation

Background

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.

Purpose

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.

Results

Compounds 1 and 3, isolated from n-hexane extract from aerial parts of B. sphenophylla, showed potent activity against parasites with EC₅₀ 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 Ca²⁺ 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.

Conclusion

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.

中文翻译：

通过生物活性引导分馏从 Baccharis sphenophylla 的正己烷提取物中分离的 Ent-kaurane 二萜靶向克氏锥虫中的酸钙体

背景

在目前的工作的生物活性引导分级ñ从地上部分提取物正己烷Baccharis sphenophylla（菊科）针对的锥鞭毛体形式的克氏锥虫进行。

目的

评估二萜类ent- kaurenoic ( 1 )、grandifloric ( 2 )的抗锥虫潜力。和15β-tiglinoyloxy- ENT -kaurenoic（3）酸，分离自Ñ从地上部分提取物正己烷B. sphenophylla，并阐明其对作用机制锥虫。

方法/研究设计

ñ从地上部分正己烷和MeOH提取物B. sphenophylla制备并引起的，分别为100％和的锥鞭毛体形式的死亡的50％锥虫。基于这些结果，该Ñ -己烷提取物进行生物活性引导分级程序，得到三个相关ENT -kaurane二萜类化合物（1-3）。基于荧光光谱分析和流式细胞术分析，研究了化合物1和3的作用机制。

结果

化合物1和3，从分离Ñ从地上部分提取物正己烷B. sphenophylla，显示出有效的活性抗寄生虫与EC _50个分别为10.6μM（SI> 18.8）和2.4μM（SI = 34.8），的值。另一方面，化合物2对锥鞭毛体无活性。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诱导质膜电位 (ΔΨp) 去极化。在用化合物3处理后，在线粒体中没有观察到实质性的改变，但是通过化合物1观察到线粒体膜电位 (ΔΨm) 的瞬时超极化。尽管由化合物1和3诱导的 ATP 水平增加，但没有记录到 ROS 和 Ca ²⁺水平的改变。然而，这两种化合物都促进了酸钙体的时间依赖性碱化，这可能导致细胞渗透失衡。化合物1 – 3 的计算机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，没有检测到锅的测定干扰化合物（PAINS）警报化合物1 - 3。