Evaluation of Anti-Trypanosoma cruzi Activity of Chemical Constituents from Baccharis sphenophylla Isolated Using High-Performance Countercurrent Chromatography.

Endemic in 21 countries, Chagas disease, also known as American Trypanosomiasis, is a neglected tropical disease (NTD) caused by the protozoan parasite Trypanosoma cruzi. The available drugs for the treatment of this disease, benznidazole and nifurtimox, are outdated and display severe side effects. Thus, the discovery of new drugs is crucial. Based on our continuous studies aiming towards the discovery of natural products with anti-T. cruzi potential, the MeOH extract from aerial parts of Baccharis sphenophylla Dusén ex. Malme (Asteraceae) displayed activity against this parasite and was subjected to high-performance countercurrent chromatography (HPCCC), to obtain one unreported syn-labdane diterpene - sphenophyllol (1) - as well as the known compounds gaudichaudol C (2), ent-kaurenoic acid (3), hispidulin (4), eupafolin (5), and one mixture of di-O-caffeoylquinic acids (6-8). Compounds 1-8 were characterized by analysis of nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. When tested against trypomastigote forms, isolated labdane diterpenes 1 and 2 displayed potent activity, with EC50 values of 20.1 µM and 2.9 µM, respectively. The mixture of chlorogenic acids 6-8, as well as the isolated flavones 4 and 5, showed significant activity against the clinically relevant amastigotes, with EC50 values of 24.9, 12.8, and 2.7 µM, respectively. Nonetheless, tested compounds 1-8 displayed no cytotoxicity against mammalian cells (CC50 > 200 µM). These results demonstrate the application of HPCCC as an important tool to isolate bioactive compounds from natural sources, including the antitrypanosomal extract from B. sphenophylla, allowing for the development of novel strategic molecular prototypes against tropical neglected diseases.

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 ent­kaurenoic (1), grandifloric (2). and 15ß-tiglinoyloxy­ent-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 ent­kaurane 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 ent­kaurane 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.