Styrylpyrone, isolated from an Amazon plant, induces cell cycle arrest and autophagy in Leishmania amazonensis.

The search for bioactive compounds against diseases is imperative and the richness of the Amazon provides a large source to be explored. Current therapies for the treatment of parasitic infections have severe side effects and low efficacy, which makes the development of an effective chemotherapy extremely important. In this study, we describe the isolation of styrylpyrone 4-methoxy-6-(11,12-methylenedioxy-trans-styryl)-2-pyrone (SP), from the Amazonian tree species, Aniba panurensis, the in vitro activity against Leishmania amazonensis promastigotes, and its in silico pharmacokinetics properties. The results showed morphological and ultrastructural alterations, cell cycle impairment, increased reactive oxygen species production, accumulation of lipid bodies and formation of autophagic vacuoles in SP-treated parasites. In silico studies revealed that the compound has a high drug-score, which is encouraging for further investigation. Our results indicate that SP is a promising drug candidate, which induces alterations in L. amazonensis leading to parasite death through cell cycle arrest and autophagy.

The extended production of UV-induced reactive oxygen species in L929 fibroblasts is attenuated by posttreatment with Arrabidaea chica through scavenging mechanisms.

Ultraviolet radiation (UVR) exposure causes various injurious effects to human skin by generating reactive oxygen species (ROS). Excessive ROS production can lead to oxidative stress which may damage cellular components like lipids and proteins and causing photoaging. The use of natural photochemopreventive agents with antioxidant properties is an important alternative to improve the effectiveness of sunscreens and reduce skin photodamage. A crude extract (CE) from the leaves of Arrabidaea chica underwent partition by a liquid-liquid method. The hexane fraction (FH), chloroform fraction (FC), and ethyl acetate fraction (FEA) were obtained. The antioxidant capacity of the CE, FH, FC, and FEA was studied in a cell-free system using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and the xanthine/luminol/xanthine oxidase system. The FC had the best antioxidant activity. We also evaluated the photochemoprotective effect of A. chica in protecting L929 fibroblasts against UV-A- and UV-B-induced cell damage. A. chica inhibited the extended production of ROS up to 3h. Posttreatment with the CE and FC attenuated UV-induced cell damage through scavenging mechanisms, including the quenching of intracellular ROS and mitochondrial O2- and preventing lipid peroxidation. These results suggest that A. chica may be a promising non-sunscreen photoprotector that can improve the effectiveness of commercial sunscreens.

C5 induces different cell death pathways in promastigotes of Leishmania amazonensis.

Leishmaniasis is a neglected infection that is caused by Leishmania protozoa, affecting millions of people worldwide, mainly in tropical and subtropical regions. This disease has different clinical forms: cutaneous, mucocutaneous, and visceral. The drugs that are currently available for the treatment of this infection have limitations, such as high toxicity, long-term treatment, and leads to drug-resistant strains. Numerous studies, in various experimental models, have sought to develop more effective and less toxic chemotherapeutic agents against leishmaniasis. In the present study, we evaluated the mechanism of cell death that is induced by n-benzyl 1-(4-methoxy)phenyl-9H-ß-carboline-3-carboxamide (C5) against Leishmania amazonensis. C5 increased reactive oxygen species production, depolarization of the mitochondrial membrane, DNA fragmentation, decrease of cell volume, lipoperoxidation, the accumulation of lipid bodies, and acidic vesicular organelles (AVOs) and caused the intense formation of autophagic compartments in L. amazonensis promastigotes. The results indicate that C5 causes L. amazonensis death through different pathways.

Vitamin K3 induces antiproliferative effect in cervical epithelial cells transformed by HPV 16 (SiHa cells) through the increase in reactive oxygen species production.

PURPOSE: Cervical cancer is characterized as an important public health problem. According to latest estimates, cancer of the cervix is the fourth most common cancer among women. Due to its high prevalence, the search for new and efficient drugs to treat this infection is continuous. The progression of HPV-associated cervical cancer involves the expression of two viral proteins, E6 and E7, which are rapidly degraded by the ubiquitin-proteasome system through the increase in reactive oxygen species generation. Vitamins are essential to human substances, participate in the regulation of metabolism, and facilitate the process of energy transfer. METHODS: Some early studies have indicated that vitamin K3 exerts antitumor activity by inducing cell death by apoptosis through an increase in the generation of reactive oxygen species. Thus, we evaluated the antiproliferative effect and a likely mechanism of action of vitamin K3 against cervical epithelial cells transformed by HPV 16 (SiHa cells) assessing the production of total ROS, the mitochondrial membrane potential, the cell morphology, the cell volume, and the cell membrane integrity. RESULTS: Our results show that vitamin K3 induces an increase in ROS production in SiHa cells, triggering biochemical and morphological events, such as depolarization of mitochondrial membrane potential and decreasing cell volume. CONCLUSION: Our data showed that vitamin K3 generates an oxidative imbalance in SiHa cells, leading to mechanisms that induce cell death by apoptosis.

Dibenzylideneacetones Are Potent Trypanocidal Compounds That Affect the Trypanosoma cruzi Redox System.

Despite ongoing efforts, the available treatments for Chagas' disease are still unsatisfactory, especially in the chronic phase of the disease. Our previous study reported the strong trypanocidal activity of the dibenzylideneacetones A3K2A1 and A3K2A3 against Trypanosoma cruzi (Z. Ud Din, T. P. Fill, F. F. de Assis, D. Lazarin-Bidóia, V. Kaplum, F. P. Garcia, C. V. Nakamura, K. T. de Oliveira, and E. Rodrigues-Filho, Bioorg Med Chem 22:1121-1127, 2014, http://dx.doi.org/10.1016/j.bmc.2013.12.020). In the present study, we investigated the mechanisms of action of these compounds that are involved in parasite death. We showed that A3K2A1 and A3K2A3 induced oxidative stress in the three parasitic forms, especially trypomastigotes, reflected by an increase in oxidant species production and depletion of the endogenous antioxidant system. This oxidative imbalance culminated in damage in essential cell structures of T. cruzi, reflected by lipid peroxidation and DNA fragmentation. Consequently, A3K2A1 and A3K2A3 induced vital alterations in T. cruzi, leading to parasite death through the three pathways, apoptosis, autophagy, and necrosis.

Trypanocidal activity of organic extracts from the Brazilian and Spanish marine sponges

Abstract Chagas' disease is a parasitic infection caused by protozoan Trypanosoma cruzi that affect millions of people worldwide. The available drugs for treatment of this infection cause serious side effects and have variable efficacy, especially in the chronic phase of the disease. In this context, natural compounds have shown great potential for the discovery of new chemotherapies for the treatment of this infection and various other diseases. In present study, we evaluated the in vitro antiprotozoal activity of five species of Brazilian and Spanish marine sponges (Condrosia reniformes, Tethya rubra, Tethya ignis, Mycale angulosa and Dysidea avara) against T. cruzi. By GC–MS data, we observed that in these extracts were present the major classes of the following compounds: hydrocarbons, terpenes, steroids and alcohols. The extracts showed activity against the three forms of this parasite and did not induce toxicity in mammalian cells. Better activities were observed with the extracts of marine sponges, C. reniformes (EC50 = 0.6 μg/ml), D. avara (EC50 = 1.1 μg/ml) and M. angulosa (EC50 = 3.8 μg/ml), against trypomastigote forms. In intracellular amastigote forms, the extract of T. ignis showed IC50 of 7.2 μg/ml and SI of 24.65. On this basis, our results indicate that these extracts can be promising chemotherapeutic agents against T. cruzi.

Mitochondrial Dysfunction Induced by N-Butyl-1-(4-Dimethylamino)Phenyl-1,2,3,4-Tetrahydro-ß-Carboline-3-Carboxamide Is Required for Cell Death of Trypanosoma cruzi.

BACKGROUND: Chagas' disease is caused by the protozoan Trypanosoma cruzi and affects thousands of people worldwide. The available treatments are unsatisfactory, and new drugs must be developed. Our group recently reported the trypanocidal activity of the synthetic compound N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxamide (C4), but the mechanism of action of this compound was unclear. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the mechanism of action of C4 against epimastigote and trypomastigote forms of T. cruzi. The results showed alterations in mitochondrial membrane potential, alterations in cell membrane integrity, an increase in the formation of reactive oxygen species, phosphatidylserine exposure, a reduction of cell volume, DNA fragmentation, and the formation of lipid inclusions. CONCLUSION/SIGNIFICANCE: These finding suggest that mitochondria are a target of C4, the dysfunction of which can lead to different pathways of cell death.

Additional evidence of the trypanocidal action of (-)-elatol on amastigote forms through the involvement of reactive oxygen species.

Chagas' disease, a vector-transmitted infectious disease, is caused by the protozoa parasite Trypanosoma cruzi. Drugs that are currently available for the treatment of this disease are unsatisfactory, making the search for new chemotherapeutic agents a priority. We recently described the trypanocidal action of (-)-elatol, extracted from the macroalga Laurencia dendroidea. However, nothing has been described about the mechanism of action of this compound on amastigotes that are involved in the chronic phase of Chagas' disease. The goal of the present study was to evaluate the effect of (-)-elatol on the formation of superoxide anions (O2•-), DNA fragmentation, and autophagy in amastigotes of T. cruzi to elucidate the possible mechanism of the trypanocidal action of (-)-elatol. Treatment of the amastigotes with (-)-elatol increased the formation of O2•- at all concentrations of (-)-elatol assayed compared with untreated parasites. Increased fluorescence was observed in parasites treated with (-)-elatol, indicating DNA fragmentation and the formation of autophagic compartments. The results suggest that the trypanocidal action of (-)-elatol might involve the induction of the autophagic and apoptotic death pathways triggered by an imbalance of the parasite's redox metabolism.

The Effects of N-Butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro- ß -carboline-3-carboxamide against Leishmania amazonensis Are Mediated by Mitochondrial Dysfunction.

Leishmaniasis is a disease that affects millions of people worldwide. The drugs that are available for the treatment of this infection exhibit high toxicity and various side effects. Several studies have focused on the development of new chemotherapeutic agents that are less toxic and more effective against trypanosomatids. We investigated the effects of N-butyl-1-(4-dimethylamino)phenyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxamide (C4) and its possible targets against L. amazonensis. The results showed morphological and ultrastructural alterations, depolarization of the mitochondrial membrane, the loss of cell membrane integrity, and an increase in the formation of mitochondrial superoxide anions in L. amazonensis treated with C4. Our results indicate that C4 is a selective antileishmanial agent, and its effects appear to be mediated by mitochondrial dysfunction.

The natural compounds piperovatine and piperlonguminine induce autophagic cell death on Trypanosoma cruzi.

The currently available treatments for Chagas disease show limited therapeutic potential and are associated with serious side effects. Our group has been attempting to find alternative drugs isolated from natural products as a potential source of pharmacological agents against Trypanosoma cruzi. Here, we demonstrate the antitrypanosomal activity of the amides piperovatine and piperlonguminine isolated from Piper ovatum against epimastigotes and intracellular amastigotes. We also investigated the mechanisms of action of these compounds on extracellular amastigote and epimastigote forms of T. cruzi. These amides showed low toxicity to LLCMK(2) mammalian cells. By using transmission and scanning electron microscopy, we observed that the compounds caused severe alterations in T. cruzi. These alterations were mainly located in plasma membrane and mitochondria. Furthermore, the study of treated parasites labeled with Rh123, PI and MDC corroborate with our TEM data. These mitochondrial dysfunctions induced by the amides might trigger biochemical alterations that lead to cell death. Altogether, our data evidence a possible autophagic process.

Trypanocidal action of (-)-elatol involves an oxidative stress triggered by mitochondria dysfunction.

Natural compounds have shown good potential for the discovery of new chemotherapeutics for the treatment of Chagas' disease. Recently, our group reported the effective trypanocidal activity of (-)-elatol, extracted from the red macroalgae Laurencia dendroidea present in the Brazilian coast against Trypanosoma cruzi. However, the mechanism of action of this compound has remained unclear. There are only hypotheses concerning its action on mitochondrial function. Here, we further investigated the mechanisms of action of (-)-elatol on trypomastigotes of T. cruzi. For this, we evaluated some biochemical alterations in trypomastigotes treated with (-)-elatol. Our results show that (-)-elatol induced depolarization of the mitochondrial membrane, an increase in the formation of mitochondrial superoxide anion and loss of cell membrane and DNA integrity. Additionally, (-)-elatol induced formation of autophagic vacuoles and a decrease in cell volume. All together, these results suggest that the trypanocidal action of (-)-elatol involves multiple events and mitochondria might be the initial target organelle. Our hypothesis is that the mitochondrial dysfunction leads to an increase of ROS production through the electron transport chain, which affects cell membrane and DNA integrity leading to different types of parasite death.

Mitochondria Superoxide Anion Production Contributes to Geranylgeraniol-Induced Death in Leishmania amazonensis.

Here we demonstrate the activity of geranylgeraniol, the major bioactive constituent from seeds of Bixa orellana, against Leishmania amazonensis. Geranylgeraniol was identified through (1)H and (13)C nuclear magnetic resonance imaging and DEPT. The compound inhibited the promastigote and intracellular amastigote forms, with IC(50) of 11 ± 1.0 and 17.5 ± 0.7 µg/mL, respectively. This compound was also more toxic to parasites than to macrophages and did not cause lysis in human blood cells. Morphological and ultrastructural changes induced by geranylgeraniol were observed in the protozoan by electronic microscopy and included mainly mitochondria alterations and an abnormal chromatin condensation in the nucleus. These alterations were confirmed by Rh 123 and TUNEL assays. Additionally, geranylgeraniol induces an increase in superoxide anion production. Collectively, our in vitro studies indicate geranylgeraniol as a selective antileishmanial that appears to be mediated by apoptosis-like cell death.

Triagem fitoquímica e avaliação das atividades antimicrobiana e citotóxica de plantas medicinais nativas da região oeste do estado do paraná / Phytochemical screening and evaluation of antimicrobial and cytotoxic activities of native medicinal plants from west region of paraná state

Muitas plantas não apresentam suas potencialidades terapêuticas comprovadas por meio de estudos científicos. Assim, o objetivo deste trabalho foi realizar um estudo fitoquímico, antimicrobiano e citotóxico das espécies vegetais Eupatorium serratum (Asteraceae), Myrcianthes pungens (Myrtaceae), Urera nitida (Urticaceae), Campomanesia xanthocarpa (Myrtaceae) e duas variedades de Psidium cattleyanum (Myrtaceae). Para tanto, as plantas foram secas e extraídas com diferentes solventes (hexano, metanol e acetato de etila) obtendo-se 18 extratos brutos. Métodos específicos foram usados para identificação de alcalóides, saponinas, flavonóides, taninos e esteróides. Para determinar as concentrações inibitória mínima (CIM) e bactericida mínima (CBM) utilizaram-se bactérias Gram positivas (Staphylococcus aureus e Micrococcus luteus) e Gram negativas (Escherichia coli e Salmonella typhi). O método de difusão em disco foi usado contra leveduras (Candida albicans and Saccharomyces cerevisiae). C. xanthocarpa, M. pungens (extratos acetato de etila e hexano) e uma variedade de P. cattleyanum (extrato acetato de etila), apresentaram CIM ? 62,5 µg/ml contra bactérias Gram-positivas, o que pode estar relacionado aos taninos encontrados nas folhas dessas plantas. O extrato hexânico de M. pungens mostrou a mais forte inibição, apresentando CBM de mesma concentração. Os extratos metanólicos de C. xanthocarpa e M. pungens mostraram boa atividade antifúngica. O ensaio citotóxico verificou o efeito hemolítico e o extrato hexânico de uma variedade de P. cattleyanum apresentou alta citotoxicidade nas concentrações testadas.

Many plants do not present its proven therapeutic potentialities through scientific studies. Thus, the aim of this work was to accomplish a phytochemical, antimicrobial and cytotoxic study of the vegetal species Eupatorium serratum (Asteraceae), Myrcianthes pungens (Myrtaceae), Urera nitida (Urticaceae), Campomanesia xanthocarpa (Myrtaceae) and two varieties of Psidium cattleyanum (Myrtaceae). The plants were dried and extracted with different solvents (hexane, methanol and ethyl acetate) resulting in 18 crude extracts. Specific methods had been used for identification of alkaloids, saponnins, flavonoids, tannins and steroids. To determine the minimum inhibitory (MIC) and bactericidal (MBC) concentrations were used Gram-positive (Staphylococcus aureus and Micrococcus luteus) and Gram negative (Escherichia coli and Salmonella typhi) bacteria. The method of disc diffusion was used against yeasts (Candida albicans and Saccharomyces cerevisiae). C. xanthocarpa, M. pungens (ethyl acetate and hexane extracts) and a variety of P. cattleyanum (ethyl acetate extract), presented MIC ? 62.5 µg/ml against Gram-positive bacteria, which may be related to tannins in the leaves of these plants. The hexanic extract of M. pungens showed the strongest inhibition, presenting MBC of equal concentration. The methanolic extracts of C. xanthocarpa and M. pungens showed good antifungal activity. The cytotoxic assay verified the hemolytic effect and the hexanic extract of a variety of P. cattleyanum presented high citotoxicity at the concentrations tested.