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

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 15 beta-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 mu M (SI > 18.8) and 2.4 mu M (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 (Delta psi p). No substantial alterations were observed in the mitochondria after treatment with compound 3, but a transient hyperpolarization of the mitochondrial membrane potential (Delta psi m) 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.

Automated summary

In this study, three ent-kaurane diterpenes isolated from Baccharis sphenophylla aerial parts showed potent activity against Trypanosoma cruzi parasites. Compound 1 induced depolarization of the plasma membrane electric potential, while compound 3 caused a transient hyperpolarization of the mitochondrial membrane potential, both contributing to an osmotic imbalance in the cell. These compounds could be interesting prototypes for the discovery of new hits against T. cruzi.