Immunomodulatory, trypanocide, and antioxidant properties of essential oil fractions of Lippia alba (Verbenaceae).

BACKGROUND: Parasite persistence, exacerbated and sustained immune response, and continuous oxidative stress have been described to contribute to the development of the cardiac manifestations in Chronic Chagas Disease. Nevertheless, there are no efficient therapies to resolve the Trypanosoma cruzi infection and prevent the disease progression. Interestingly, trypanocide, antioxidant, and immunodulatory properties have been reported separately for some major terpenes, as citral (neral plus geranial), limonene, and caryophyllene oxide, presents in essential oils (EO) extracted from two chemotypes (Citral and Carvone) of Lippia alba. The aim of this study was to obtain L. alba essential oil fractions enriched with the aforementioned bioactive terpenes and to evaluate the impact of these therapies on trypanocide, oxidative stress, mitochondrial bioenergetics, genotoxicity, and inflammatory markers on T. cruzi-infected macrophages. METHODS: T. cruzi-infected J774A.1 macrophage were treated with limonene-enriched (ACT1) and citral/caryophyllene oxide-enriched (ACT2) essential oils fractions derived from Carvone and Citral-L. alba chemotypes, respectively. RESULTS: ACT1 (IC50 = 45 ± 1.7 µg/mL) and ACT2 (IC50 = 80 ± 1.9 µg/mL) exhibit similar trypanocidal effects to Benznidazole (BZN) (IC50 = 48 ± 2.5 µg/mL), against amastigotes. Synergistic antiparasitic activity was observed when ACT1 was combined with BZN (∑FIC = 0.52 ± 0.13 µg/mL) or ACT2 (∑FIC = 0.46 ± 1.7 µg/mL). ACT1 also decreased the oxidative stress, mitochondrial metabolism, and genotoxicity of the therapies. The ACT1 + ACT2 and ACT1 + BZN experimental treatments reduced the pro-inflammatory cytokines (IFN-γ, IL-2, and TNF-α) and increased the anti-inflammatory cytokines (IL-4 and IL-10). CONCLUSION: Due to its highly trypanocidal and immunomodulatory properties, ACT1 (whether alone or in combination with BZN or ACT2) represents a promising L. alba essential oil fraction for further studies in drug development towards the Chagas disease control.

Apoptosis-like cell death upon kinetoplastid induction by compounds isolated from the brown algae Dictyota spiralis.

BACKGROUND: The in vitro activity of the brown seaweed Dictyota spiralis against both Leishmania amazonensis and Trypanosoma cruzi was evaluated in a previous study. Processing by bio-guided fractionation resulted in the isolation of three active compounds, classified as diterpenes. In the present study, we performed several assays to detect clinical features associated to cell death in L. amazonensis and T. cruzi with the aim to elucidate the mechanism of action of these compounds on parasitic cells. METHODS: The aims of the experiments were to detect and evaluate specific events involved in apoptosis-like cell death in the kinetoplastid, including DNA condensation, accumulation of reactive oxygen species and changes in ATP concentration, cell permeability and mitochondrial membrane potential, respectively, in treated cells. RESULTS: The results demonstrated that the three isolated diterpenes could inhibit the tested parasites by inducing an apoptosis-like cell death. CONCLUSIONS: These results encourage further investigation on the isolated compounds as potential drug candidates against both L. amazonensis and T. cruzi.

Induction of programmed cell death in Trypanosoma cruzi by Lippia alba essential oils and their major and synergistic terpenes (citral, limonene and caryophyllene oxide).

BACKGROUND: Chagas Disease caused by Trypanosoma cruzi infection, is one of the most important neglected tropical diseases (NTD), without an effective therapy for the successful parasite eradication or for the blocking of the disease's progression, in its advanced stages. Due to their low toxicity, wide pharmacologic spectrum, and potential synergies, medicinal plants as Lippia alba, offer a promising reserve of bioactive molecules. The principal goal of this work is to characterize the inhibitory properties and cellular effects of the Citral and Carvone L. alba chemotype essential oils (EOs) and their main bioactive terpenes (and the synergies among them) on T. cruzi forms. METHODS: Twelve L. alba EOs, produced under diverse environmental conditions, were extracted by microwave assisted hydrodistillation, and chemically characterized using gas chromatography coupled mass spectrometry. Trypanocidal activity and cytotoxicity were determined for each oil, and their major compounds, on epimastigotes (Epi), trypomastigotes (Tryp), amastigotes (Amas), and Vero cells. Pharmacologic interactions were defined by a matrix of combinations among the most trypanocidal terpenes (limonene, carvone; citral and caryophyllene oxide). The treated cell phenotype was assessed by fluorescent and optic microscopy, flow cytometry, and DNA electrophoresis assays. RESULTS: The L. alba EOs displayed significant differences in their chemical composition and trypanocidal performance (p = 0.0001). Citral chemotype oils were more trypanocidal than Carvone EOs, with Inhibitory Concentration 50 (IC50) of 14 ± 1.5 µg/mL, 22 ± 1.4 µg/mL and 74 ± 4.4 µg/mL, on Epi, Tryp and Amas, respectively. Limonene exhibited synergistic interaction with citral, caryophyllene oxide and Benznidazole (decreasing by 17 times its IC50) and was the most effective and selective treatment. The cellular analysis suggested that these oils or their bioactive terpenes (citral, caryophyllene oxide and limonene) could be inducing T. cruzi cell death by an apoptotic-like mechanism. CONCLUSIONS: EOs extracted from L. alba Citral chemotype demonstrated significant trypanocidal activity on the three forms of T. cruzi studied, and their composition and trypanocidal performance were influenced by production parameters. Citral, caryophyllene oxide, and limonene showed a possible induction of an apoptotic-like phenotype. The best selective anti-T. cruzi activity was achieved by limonene, the effects of which were also synergic with citral, caryophyllene oxide and benznidazole.

Selenium-containing analogues of WC-9 are extremely potent inhibitors of Trypanosoma cruzi proliferation.

The obligate intracellular parasite, Trypanosoma cruzi is the etiologic agent of Chagas disease or American trypanosomiasis, which is the most prevalent parasitic disease in the Americas. The present chemotherapy to control this illness is still deficient particularly in the chronic stage of the disease. The ergosterol biosynthesis pathway has received much attention as a molecular target for the development of new drugs for Chagas disease. Especially, inhibitors of the enzymatic activity of squalene synthase were shown to be effective compounds on T. cruzi proliferation in in vitro assays. In the present study we designed, synthesized and evaluated the effect of a number of isosteric analogues of WC-9 (4-phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, on T. cruzi growth in tissue culture cells. The selenium-containing derivatives turned out to be extremely potent inhibitors of T. cruzi growth. Certainly, 3-phenoxyphenoxyethyl, 4-phenoxyphenoxyethyl, 4-(3-fluorophenoxy)phenoxyethyl, 3-(3-fluorophenoxy)phenoxyethyl selenocyanates and (±)-5-phenoxy-2-(selenocyanatomethyl)-2,3-dihydrobenzofuran arose as relevant members of this family of compounds, which exhibited effective ED50 values of 0.084 µM, 0.11 µM, 0.083, µM, 0.085, and 0.075 µM, respectively. The results indicate that compounds bearing the selenocyanate moiety are at least two orders of magnitude more potent than the corresponding skeleton counterpart bearing the thiocyanate group. Surprisingly, these compounds exhibited excellent selectively index values ranging from 900 to 1800 making these molecules promising candidates as antiparasitic agents.

An image-based algorithm for precise and accurate high throughput assessment of drug activity against the human parasite Trypanosoma cruzi.

We present a customized high content (image-based) and high throughput screening algorithm for the quantification of Trypanosoma cruzi infection in host cells. Based solely on DNA staining and single-channel images, the algorithm precisely segments and identifies the nuclei and cytoplasm of mammalian host cells as well as the intracellular parasites infecting the cells. The algorithm outputs statistical parameters including the total number of cells, number of infected cells and the total number of parasites per image, the average number of parasites per infected cell, and the infection ratio (defined as the number of infected cells divided by the total number of cells). Accurate and precise estimation of these parameters allow for both quantification of compound activity against parasites, as well as the compound cytotoxicity, thus eliminating the need for an additional toxicity-assay, hereby reducing screening costs significantly. We validate the performance of the algorithm using two known drugs against T.cruzi: Benznidazole and Nifurtimox. Also, we have checked the performance of the cell detection with manual inspection of the images. Finally, from the titration of the two compounds, we confirm that the algorithm provides the expected half maximal effective concentration (EC50) of the anti-T. cruzi activity.

New oxirane derivatives of 1,4-naphthoquinones and their evaluation against T. cruzi epimastigote forms.

New oxirane derivatives were synthesized using six naphthoquinones as the starting materials. Our biological results showed that these oxiranes acted as trypanocidal agents against Trypanosoma cruzi with minimal cytotoxicity in the VERO cell line compared to naphthoquinones. In particular, oxirane derivative 14 showed low cytotoxicity in a mammalian cell line and exhibited better activity against epimastigote forms of T.cruzi than the current drug used to treat Chagas disease, benznidazole.

Indomethacin amides as a novel molecular scaffold for targeting Trypanosoma cruzi sterol 14alpha-demethylase.

Trypanosoma cruzi (TC) causes Chagas disease, which in its chronic stage remains incurable. We have shown recently that specific inhibition of TC sterol 14alpha-demethylase (TCCYP51) with imidazole derivatives is effective in killing both extracellular and intracellular human stages of TC. An alternative set of TCCYP51 inhibitors has been identified using optical high throughput screening followed by web-database search for similar structures. The best TCCYP51 inhibitor from this search was found to have structural similarity to a class of cyclooxygenase-2-selective inhibitors, the indomethacin-amides. A number of indomethacin-amides were found to bind to TCCYP51, inhibit its activity in vitro, and produce strong antiparasitic effects in the cultured TC cells. Analysis of TC sterol composition indicated that the mode of action of the compounds is by inhibition of sterol biosynthesis in the parasite.

L-arginine-dependent suppression of apoptosis in Trypanosoma cruzi: contribution of the nitric oxide and polyamine pathways.

Until recently, a capacity for apoptosis and synthesis of nitric oxide *NO) were viewed as exclusive to multicellular organisms. The existence of these processes in unicellular parasites was recently described, with their biological significance remaining to be elucidated. We have evaluated L-arginine metabolism in Trypanosoma cruzi in the context of human serum-induced apoptotic death. Apoptosis was evidenced by the induction of DNA fragmentation and the inhibition of [3H]thymidine incorporation, which were inhibited by the caspase inhibitor Ac-Asp-Glu-Val-aspartic acid aldehyde (DEVD-CHO). In T. cruzi exposed to death stimuli, supplementation with L-arginine inhibited DNA fragmentation, restored [3H]thymidine incorporation, and augmented parasite *NO production. These effects were inhibited by the *NO synthase inhibitor N(omega)-nitroarginine methyl ester (L-NAME). Exogenous *NO limited DNA fragmentation but did not restore proliferation rates. Because L-arginine is also a substrate for arginine decarboxylase (ADC), and its product agmatine is a precursor for polyamine synthesis, we evaluated the contribution of polyamines to limiting apoptosis. Addition of agmatine, putrescine, and the polyamines spermine and spermidine to T. cruzi sustained parasite proliferation and inhibited DNA fragmentation. Also, the ADC inhibitor difluoromethylarginine inhibited L-arginine-dependent restoration of parasite replication rates, while the protection from DNA fragmentation persisted. In aggregate, these results indicate that T. cruzi epimastigotes can undergo programmed cell death that can be inhibited by L-arginine by means of (i) a *NO synthase-dependent *NO production that suppresses apoptosis and (ii) an ADC-dependent production of polyamines that support parasite proliferation.

In vitro antiproliferative effects and mechanism of action of the new triazole derivative UR-9825 against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi.

We describe the in vitro antiproliferative effects of the new triazole derivative UR-9825 against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease in Latin America. The compound was found to be extremely active against the cultured (epimastigote) form of the parasite, equivalent to that present in the reduviid vector, with a MIC of 30 nM, a concentration 33-fold lower than that required with the reference compound ketoconazole. At that MIC, growth arrest coincided with depletion of the parasite's 4,14-desmethyl endogenous sterols (ergosterol, 24-ethylcholesta-5,7,22-trien-3b-ol, and precursors) and their replacement by methylated sterols (lanosterol, 24-methylenedihydrolanosterol, and obtusifoliol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of UR-9825 was inhibition of the parasite's sterol C14alpha demethylase, as seen with other azole derivatives. The phospholipid composition of growth-arrested epimastigotes was also altered, when compared to controls, with a significant increase in the content of phosphatidylethanolamine and phosphatidylserine and a concomitant reduction of the content of phosphatidylcholine. The clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37 degrees C, was even more sensitive to UR-9825, with a MIC of 10 nM, comparable to that for ketoconazole. The results showed that UR-9825 is among the most potent azole derivatives tested against this parasite and support in vivo studies with this compound.

[Activity of Dipernoids isolated from Azorella compacta (Llareta) on Trypanosoma cruzi amastigotes]. / Actividad de diterpenoides de Azorella compacta (Llareta) sobre amastigotes de Trypanosoma cruzi.

The trypanocidad activity against amastigote forms of SPA-14, Tulahuen and G strains and CL Brener clone of Trypanosoma cruzi of diterpenoids isolated from Azorella compacta, Phil. (Llareta), a plant with ethnomedicinal prestige from prespanish age, was investigated. Amastigocidal activity was shown in azorellanol (2), diterpene isolated by first time, with an inhibitory concentration 50 (IC50) that varied between 60 M (CL Brener clone) and 84 M (SPA-14 strain), and in mulin -11,13 -dien-20-oico acid (5) with IC50 between 41 microM (G strain) and 87 mM (CL Brener clone). The cytotoxicity levels of both compounds against Hela and Vero cells and macrophages J144 are lower than nifurtimox and similar to gentian violet.

Two distinct groups of mucin-like genes are differentially expressed in the developmental stages of Trypanosoma cruzi.

Sialic acid acceptors of Trypanosoma cruzi are abundant mucin-like glycoproteins linked to the parasite membrane by a glycosylphosphatidyl inositol (GPI) anchor. They are heterogeneous and variable in different parasite stages. The protein portion of these mucins contains many threonine residues, and is thought to be encoded by a heterogeneous gene family. To investigate whether the high degree of heterogeneity in the mucin gene family is responsible for the diversity of mucins expressed on the parasite surface, we have studied the expression of mucin genes in several developmental stages of T. cruzi. We have found that mucins are expressed in all parasite stages. By using conserved sequences at 3' end of translated sequences of the gene family and the splice leader sequence, we have isolated 120 mucin-like cDNAs by RT-PCR from epimastigote and trypomastigote mRNAs. All transcribed genes contain conserved 5' and 3' regions, which code for the signal peptide, the sequence for GPI anchor addition, and a conserved domain rich in threonine residues. The internal portions of these genes are highly variable in size and sequence, and can be grouped in two major categories. One group contains KP(1-2)T(6-8) repeats, a motif found in mammalian mucins in the central region. This group is expressed preferentially in the trypomastigote forms ready to be released from the infected mammalian cell. The other has highly variable sequences in the central portion, and is expressed in all parasite stages. Because the number of synonymous substitutions is equivalent to the non-synonymous substitutions in the second group, they are probably evolving neutrally. On the other hand, the KP(1-2)T(6-8) containing genes have more synonymous substitutions and are most likely under a strong selective pressure. We propose that the group of KP(1-2)T(6-8) motif corresponds to the highly glycosylated mucins of the trypomastigote stages. In the other group proteolysis may remove the central domain yielding small mucins, such as the mucins found in insect derived stages of T. cruzi.