Tânia Ueda-Nakamura

Recent advances in leishmaniasis treatment

About 1.5 million new cases of cutaneous leishmaniasis and 500,000 new cases of visceral leishmaniasis occur each year around the world. For over half a century, the clinical forms of the disease have been treated almost exclusively with pentavalent antimonial compounds. In this review, we describe the arsenal available for treating Leishmania infections, as well as recent advances from research on plants and synthetic compounds as source drugs for treating the disease. We also review some new drug-delivery systems for the development of novel chemotherapeutics. We observe that the pharmaceutical industry should employ its modern technologies, which could lead to better use of plants and their extracts, as well as to the development of synthetic and semi-synthetic compounds. New studies have highlighted some biopharmaceutical technologies in the design of the delivery strategy, such as nanoparticles, liposomes, cochleates, and non-specific lipid transfer proteins. These observations serve as a basis to indicate novel routes for the development and design of effective anti-Leishmania drugs.

Antimicrobial activity of Brazilian copaiba oils obtained from different species of the Copaifera genus

The antimicrobial activity of copaiba oils was tested against Gram-positive and Gram-negative bacteria, yeast, and dermatophytes. Oils obtained from Copaifera martii, Copaifera officinalis, and Copaifera reticulata (collected in the state of Acre) were active against Gram-positive species (Staphylococcus aureus, methicillin-resistant S. aureus, Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus faecalis) with minimum inhibitory concentrations ranging from 31.3-62.5 microg/ml. The oils showed bactericidal activity, decreasing the viability of these Gram-positive bacteria within 3 h. Moderate activity was observed against dermatophyte fungi (Trichophyton rubrum and Microsporum canis). The oils showed no activity against Gram-negative bacteria and yeast. Scannning electron microscopy of S. aureus treated with resin oil from C. martii revealed lysis of the bacteria, causing cellular agglomerates. Transmission electron microscopy revealed disruption and damage to the cell wall, resulting in the release of cytoplasmic compounds, alterations in morphology, and a decrease in cell volume, indicating that copaiba oil may affect the cell wall.

Potent antifungal activity of extracts and pure compound isolated from pomegranate peels and synergism with fluconazole against Candida albicans.

Activity-guided repeated fractionation of crude hydro alcoholic extract prepared from the fruit peel of Punica granatum on a silica-gel column yielded a compound that exhibited strong antifungal activity against Candida spp. Based on spectral analyses, the compound was identified as punicalagin. Punicalagin showed strong activity against Candida albicans and Candida parapsilosis, with MICs of 3.9 and 1.9 microg/ml, respectively. The combination of punicalagin and fluconazole showed a synergistic interaction. MIC for fluconazole decreased twofold when combined with the extract. The FIC index was 0.25. The synergism observed in disk-diffusion and checkerboard assays was confirmed in time-kill curves. The effect of punicalagin on the morphology and ultrastructure in treated yeast cells was examined by scanning and transmission electron microscopy. An irregular budding pattern and pseudohyphae were seen in treated yeasts. By transmission electron microscopy, treated cells showed a thickened cell wall, changes in the space between cell wall and the plasma membrane, vacuoles, and a reduction in cytoplasmic content. Since the punicalagin concentration effective in vitro is achievable in vivo, the combination of this agent with fluconazole represents an attractive prospect for the development of new management strategies for candidiasis, and should be investigated further in in vivo models.

Effect of Brazilian copaiba oils on Leishmania amazonensis.

ETHNOPHARMACOLOGICAL RELEVANCE Copaiba oil has been used in folk medicine since the 19th century. The use of copaiba oils to treat leishmaniasis is cited in several ethnopharmacological studies. Nevertheless, the potential antileishmania of copaiba oils had not been studied. AIM OF THE STUDY Eight different kinds of Brazilian copaiba oils were screened for antileishmanial activity. MATERIALS AND METHODS The antiproliferative effect of copaiba oil on promastigote and amastigote axenic were determined. To determine the survival index peritoneal macrophage were infected with promastigotes of Leishmania amazonensis and treated with copaiba oil. The cytotoxic effect of copaiba oil was assessed on macrophage strain J774G8 by assay of sulforhodamine B. RESULTS Copaiba oils showed variable levels of activity against promastigote forms with IC(50) values in the range between 5 and 22microg/mL. The most active oil was that from Copaifera reticulata (collected in Pará State, Brazil) with IC(50) values of 5, 15, and 20microg/mL for promastigote, axenic amastigote and intracellular amastigote forms, respectively. Amphotericin B showed IC(50) of 0.058 and 0.231microg/mL against promastigote and amastigote forms, respectively. Cytotoxicity assay showed that this copaiba oil obtained from Copaifera reticulata showed low cytotoxicity against J774G8 macrophages. CONCLUSION Copaiba oils showed significant activity against the parasite Leishmania amazonensis.

Effects of medicinal plant extracts on growth of Leishmania (L.) amazonensis and Trypanosoma cruzi

This study describes the screening of extracts obtained from 19 species of plants used in Brazilian traditional medicine for treatment of a variety of diseases. The extracts were tested against axenic amastigote and promastigote forms of Leishmania (L.) amazonensis, and epimastigote forms of Trypanosoma cruzi in vitro at a concentration of 100 mg/ml. Baccharis trimera, Cymbopogon citratus, Matricaria chamomilla, Mikania glomerata, Ocimum gratissimum, Piper regnellii, Prunus domestica, Psidium guajava, Sambucus canadensis, Stryphnodendron adstringens, Tanacetum parthenium, and Tanacetum vulgare showed significant effects against one or both parasites, with a percentage of growth inhibition between 49.5 and 99%. The extracts showed no cytotoxic effect on sheep erythrocytes. These medicinal plants may be sources of new compounds that are clinically active against L. amazonensis and T. cruzi.

Comparison of the bacteriological quality of tap water and bottled mineral water.

The bacteriological quality of tap water from municipal water supplies, 20-L bottles of mineral water from water dispensers and samples collected from new 20-L bottles of mineral water were comparatively studied. Total coliforms, termotolerant coliforms, Escherichia coli, fecal streptococci, Pseudomonas aeruginosa, Staphylococcus spp. and heterotrophic plate count were enumerated. The results showed that 36.4% of the tap water samples from municipal water systems and 76.6% of the 20-L bottles of mineral water from water dispensers were contaminated by at least one coliform or indicator bacterium and/or at least one pathogenic bacterium. The bacteriological quality of municipal tap water is superior when compared with the 20-L bottles of mineral water collected from water dispensers and samples collected from new 20-L bottles of mineral water before installation in the dispensers. This highlights the need for an improved surveillance system for the bottled water industry. For the municipal water systems, it is recommended to perform the Pseudomonas enumeration periodically, in addition to the routine data collected by most systems.

In vitro antifungal activity of extracts and neolignans from Piper regnellii against dermatophytes

The present study was designated to evaluate the in vitro antidermatophyte activity of extracts from leaves of Piper regnellii as well as of the bioactivity-directed isolation of neolignans. The antifungal assay was performed by microdilution techniques. The hydroalcoholic extract of Piper regnellii leaves presented a strong activity against the dermatophyte fungi Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Microsporum gypseum with MICs of 15.62, 15.62, 15.62 and 62.5 microg/ml, respectively. On light microscopy and scanning electron microscopy of nail fragments not exposed to hydroalcoholic extract of Piper regnelli leaves, well-formed and extensive mycelial growth was seen. On nail fragments exposed to hydroalcoholic extract at concentrations more than 1.2mg/ml and then inoculated with spore suspension, growth was not seen. The hydroalcoholic extract was fractionated on silica gel in to nine fractions. The active chloroform fraction was lyophilized and chromatographed by column chromatography on silica gel. Structures were established by comparison with literature data and identified as eupomatenoid-3 and eupomatenoid-5. The pure compounds showed strong activity on Trichophyton rubrum with MIC of 50 and 6.2 microg/ml, respectively. Comparing the activity of the active chloroform fraction obtained from hydroalcoholic crude extract with that of isolated compound eupomatenoid-5, it is clear that this showed the same results against Trichophyton rubrum. The results showed that the plant could be explored for possible antifungal agents and provides preliminary scientific validation for the traditional medicinal use of this plant.

Evaluation of antileishmanial activity of eupomatenoid-5, a compound isolated from leaves of Piper regnellii var. pallescens.

Infection with Leishmania spp. causes a disease with multifaceted clinical manifestations in humans. The treatment for leishmaniasis is dependent on a limited range of drugs. Here we investigated the antileishmanial activity of eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. We showed that eupomatenoid-5 had a dose-dependent activity during 72h of treatment, exhibiting IC(50) of 9.0microg/mL and 13.0microg/mL for promastigote and axenic amastigote forms, respectively, and IC(50) of 5.0microg/mL for intracellular amastigote forms of Leishmania amazonensis. When L. amazonensis was treated with eupomatenoid-5, it underwent considerable ultrastructural alterations, as shown by transmission electron microscopy. Among the alterations was the appearance of intense exocytic activity in the region of the flagellar pocket, myelin-like figures, and vacuoles in the cytoplasm of parasites treated with 9.0microg/mL. Cells treated with 25.0microg/mL showed a very large structure, apparently an extension of the endoplasmic reticulum. Also, mitochondrial swelling was detected at this concentration, indicating damage and significant change in this organelle. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa and it is not toxic to macrophages. Our studies indicated that eupomatenoid-5 might be a potential new drug for the treatment of leishmaniasis, because this compound displays interesting antileishmanial activity in vitro against promastigote, axenic amastigote, and intracellular amastigote forms of L. amazonensis.

Biological activity of 1,2,3,4-tetrahydro-β-carboline-3-carboxamides against Trypanosoma cruzi

Several beta-carboline compounds were evaluated for in vitro trypanocidal activity against Trypanosoma cruzi and their potential toxic effects was also assessed. beta-Carboline derivative 4 showed good activity against epimastigote, trypomastigote, and amastigote forms of T. cruzi, with a dose-dependent inhibitory effect. It showed an IC(50) of 14.9 microM against the epimastigote form and an EC(50) of 45 microM and 33 microM against trypomastigote and amastigote forms, respectively. Additionally, 4 was able to be active on mammalian cell-protozoan interaction, reducing the number of infected cells and the number of internalized parasites. The compound showed low cytotoxicity, with a selective index 31 times higher to the parasite than for mammalian cells. In human red-blood cells beta-Carboline 4 at 14.9 microM not caused haemolysis. Observed at electron microscopy 4-treated epimastigotes showed abnormal swelling of the mitochondrion, a diffuse kinetoplast, and distortions of the parasite cell body. The present data support the potential effect of this class of compounds against T. cruzi and encourage further experiments in vitro to evaluate the action mechanism of this drug and also with in vivo models.

Antimicrobial and cytotoxic activities of medicinal plants of the Brazilian cerrado, using Brazilian cachaça as extractor liquid

ETHNOPHARMACOLOGICAL IMPORTANCE Many species of plants in the Brazilian cerrado (savanna) are widely used in ethnomedicine. However, the safety and effectiveness of medicinal plants used in communities with little or no access to manufactured drugs should be evaluated. AIM OF THE STUDY Evaluate the antimicrobial and cytotoxic activities of extracts from eight plant species, obtained using Brazilian cachaça as the extractor liquid. MATERIALS AND METHODS The extracts were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida parapsilosis, promastigote forms of Leishmania amazonensis, and poliovirus. In addition, cytotoxic activity was assayed in Vero cells and in human erythrocytes. RESULTS The plant species Curatella americana, Sclerolobium aureum, and Plathymenia reticulata showed the best activity against yeasts, especially the crude extract of C. americana and its ethyl-acetate fraction. Kielmeyera lathrophyton showed a minimum inhibitory concentration of 250 μg/ml against S. aureus, and was inactive against gram-negative bacteria. The extract obtained from Annona coriacea showed the best activity against the promastigote forms of Leishmania amazonensis (IC(50)=175 μg/ml). Only C. americana showed potential for antipoliovirus activity. The concentrations of the crude extracts that showed toxicity to VERO cells had CC(50) between 31 and 470 μg/ml, and the lyophilized Brazilian cachaça showed a CC(50) of 307 μg/ml. None of the extracts showed toxicity against human erythrocytes. CONCLUSIONS Among the plant species studied, C. americana proved to be effective against microorganisms, especially as an antifungal. The results will help in the search for alternative drugs to be used in pharmacotherapy, and will contribute to establish safe and effective use of phytomedicines in the treatment of infectious diseases.