Ketoconazole-loaded poly-(lactic acid) nanoparticles: Characterization and improvement of antifungal efficacy in vitro against Candida and dermatophytes.

OBJECTIVE: In order to improve the effect of ketoconazole, poly-lactic acid (PLA) nanoparticles containing ketoconazole were prepared, characterized and tested against dermatophytes and Candida spp planktonic and biofilm cells. METHODS: The ketoconazole-PLA nanoparticles obtained by nanoprecipitation were characterized using dynamic light scattering, scanning electron microscopy, transmission electron microscopy, and differential scanning calorimetry. In addition, quantification of encapsulated ketoconazole and the in vitro release profile were determined. Antifungal susceptibility tests against dermatophytes Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum and yeasts Candida albicans, C. dubliniensis, C. krusei, C. parapsilosis, and C. tropicalis were performed. RESULTS: Spherical nanoparticles, with a mean diameter of 188.5nm and an encapsulation efficiency of 45% ketoconazole, were obtained. The nanoparticles containing ketoconazole had superior antifungal activity against all tested fungi strains than free ketoconazole. Inhibition of yeast biofilm formation was also achieved. CONCLUSION: Ketoconazole-PLA nanoparticles resulted in better antifungal activity of ketoconazole nanoparticles than free drug against dermatophytes and Candida species, indicating a promising tool for the development of therapeutic strategies.

Preparation, characterization and antidermatophytic activity of free- and microencapsulated cinnamon essential oil.

Essential oils (EO) are effective natural antimicrobials but are susceptible to oxidation. Microencapsulation improves EO stability, reduces toxicity, and controls release. The aim of this study was preparation, characterization and antidermatophytic activity of free and microencapsulated cinnamon essential oil (MP). MP were prepared by the spray drying method and the success of MP encapsulation was confirmed by UV-vis spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. The antifungal effect of EO and MP was evaluated by the broth microdilution method against Microsporum gypseum and Trichophyton mentagrophytes. The checkerboard method was used to assess synergistic interactions. Fluorescence microscopy and scanning electron microscopy were used to investigate the inhibition of hyphal growth by EO and MP. A cytotoxic assay was performed using the VERO cell line. Microencapsulated cinnamon essential oil was found to be micrometric, with a round, regular structure. The minimum inhibitory concentration of EO was found to be between 125-250µg/mL, while that of MP was 220.5-440.5µg/mL. EO was synergistic with fluconazole while microencapsulated oil was less cytotoxic than EO.

N-butyl-[1-(4-methoxy)phenyl-9H-ß-carboline]-3-carboxamide prevents cytokinesis in Leishmania amazonensis.

Leishmaniasis, a complex of diseases caused by protozoa of the genus Leishmania, is endemic in 98 countries, affecting approximately 12 million people worldwide. Current treatments for leishmaniasis have many disadvantages, such as toxicity, high costs, and prolonged treatment, making the development of new treatment alternatives highly relevant. Several studies have verified the antileishmanial activity of ß-carboline compounds. In the present study, we investigated the in vitro antileishmanial activity of N-butyl-[1-(4-methoxy)phenyl-9H-ß-carboline]-3-carboxamide (ß-CB) against Leishmania amazonensis. The compound was active against promastigote, axenic amastigote, and intracellular amastigote forms of L. amazonensis, exhibiting high selectivity for the parasite. Moreover, ß-CB did not exhibit hemolytic or mutagenic potential. Promastigotes treated with the alkaloid presented rounding of the body cell, cell membrane projections, an increase in the number of promastigotes presenting two flagella, and parasites of abnormal phenotype, with three or more flagella and/or nuclei. Furthermore, we observed an increase in the subpopulation of cells in the G2/M stage of the cell cycle. Altogether, these results suggest that ß-CB likely prevents cytokinesis, although it does not interfere with the duplication of cell structures. We also verified an increase in O2(·-) production and the accumulation of lipid storage bodies. Cell membrane integrity was maintained, in addition to the absence of phosphatidylserine externalization, DNA fragmentation, and autophagosomes. Although the possibility of an apoptotic process cannot be discarded, ß-CB likely exerts its antileishmanial activity through a cytostatic effect, thus preventing cellular proliferation.

Further evidence of the trypanocidal action of eupomatenoid-5: confirmation of involvement of reactive oxygen species and mitochondria owing to a reduction in trypanothione reductase activity.

Our group assays natural products that are less toxic and more effective than available nitroheterocycles as promising therapeutic options for patients with Chagas disease. Our previous study reported the trypanocidal activity of eupomatenoid-5, a neolignan isolated from the leaves of Piper regnellii var. pallescens, against the three main parasitic forms of Trypanosoma cruzi. The present study further characterizes the biochemical and morphological alterations induced by this compound to elucidate the mechanisms of action involved in the cell death of T. cruzi. We show that eupomatenoid-5 induced oxidative imbalance in the three parasitic forms, especially trypomastigotes, reflected by a decrease in the activity of trypanothione reductase and increase in the formation of reactive oxygen species (ROS). A reduction of mitochondrial membrane potential was then triggered, further impairing the cell redox system through the production of more ROS and reactive nitrogen species. Altogether, these effects led to oxidative stress, reflected by lipid peroxidation and DNA fragmentation. These alterations are key events in the induction of parasite death through various pathways, including apoptosis, necrosis, and autophagy.

Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis.

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD50 of 0.9 µM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.

Beta-carboline-3-carboxamide derivatives as promising antileishmanial agents.

Leishmaniasis has an overwhelming impact on global public health especially in tropical and subtropical countries and the currently available antileishmanial drugs have serious side effects and low efficacy. Natural and synthetic compounds have been tested in the past few years against Leishmania and the beta-carboline class of compounds have shown great results in antiparasitic chemotherapy. In the present study, three 1-substituted beta-carboline-3-carboxamides (3-5) and 1-substituted beta-carboline-3-carboxylic acid (2) were synthesized and screened for in vitro activity against L. amazonensis. Compound 5 (N-benzyl 1-(4-methoxy)phenyl-9H-beta-carboline-3-carboxamide) had the best activity against promastigote and axenic amastigote forms with IC(50) of 2·6 and 1·0 µM, respectively. Its CC(50) on macrophages cell line was higher than 2457·0 µM with an SI ratio of 930·2. Against intracellular amastigote forms, it had a dose-dependent relationship with a 50% growth inhibitory concentration of 1·0 µM. Through morphological and ultrastructure analysis of promastigote forms treated with compound 5, alterations on cell shape and number of flagella and nuclear membrane damage were observed. For this, compound 5 supports the idea for more in vitro and in vivo studies.

Antileishmanial activity of an essential oil from the leaves and flowers of Achillea millefolium.

An essential oil was recently extracted from the leaves and flowers of yarrow (Achillea millefolium) and tested for in-vitro activity against Leishmania amazonensis and murine macrophages (i.e. the J774G8 cell line). The median inhibitory concentration (IC(50)) against L. amazonensis promastigotes was 7.8 µg/ml whereas the survival of amastigotes of this pathogen, within peritoneal murine macrophages, was halved by treatment with the oil at 6.5 µg/ml. The mean value for the median cytotoxic concentration of the oil, measured against adherent (uninfected) J774G8 macrophages, was 72.0 µg/ml (i.e. 9.2 and 11.0 times higher, respectively, than the IC(50) against the promastigotes and intracellular amastigotes). Scanning electron microscopy revealed that the oil caused morphological changes in the treated parasites, including alterations in their shape and size. In transmission electron microscopy, promastigotes treated with the oil (at the IC(50) of 7.8 µg/ml) showed various ultrastructural alterations, including changes in the flagellar membrane, abnormal membrane structures, rupture of the plasma membrane, atypical vacuoles, myelin-like figures, and vesicles that resembled autophagic vacuoles.

In vitro anti-trypanosomal activity of elatol isolated from red seaweed Laurencia dendroidea.

SUMMARY: Chagas' disease is a debilitating but comparatively neglected illness that affects about 15 million people. There is an urgent need to develop new, more effective, and less-toxic compounds. In this study, we assessed the in vitro anti-trypanosomal activity of the sesquiterpene elatol from the Brazilian red seaweed Laurencia dendroidea. We used electron microscopy to evaluate the effect of elatol on the morphology and ultrastructure of the parasite. Elatol showed a dose-dependent effect against the epimastigote, trypomastigote, and amastigote forms, with IC50 values of 45.4, 1.38, and 1.01 microm, respectively. Observation of treated intracellular amastigotes by light microscopy demonstrated a total elimination of the infection at a dose of 3.0 microm. In addition, the compound did not affect the red blood cells, and the CC50 value for LLCMK2 cells was 27.0 microm. Transmission and scanning electron micrographs showed aberrant-shaped cells and breaks in the plasma membrane, prominent swollen mitochondria, and extensive formation of cytoplasmic vacuoles in all the forms. This is the first report of the anti-trypanosomal effect of the sesquiterpene elatol.

Antiviral activity and mode of action of a peptide isolated from Sorghum bicolor.

In this paper, we describe the purification of an antiviral peptide from seeds of Sorghum bicolor L. by a procedure that included gel filtration, ion exchange, and high-performance liquid chromatography (HPLC) in a reversed-phase column. Its molecular weight, determined by chromatographic mobility on the Shim-pack DIOL-150 gel permeation column in HPLC, was found to be 2000Da. The peptide designated 2kD peptide strongly inhibited the replication of herpes simplex virus type 1 (HSV-1), dose-dependently, at 40-90% of the control level, after incubation with 10-50 microM of the peptide, with EC(50) and EC(90) values of 6.25 and 15.25 microM, respectively. The IC(50) value of the 2kD peptide against Vero cells was 250 microM. Pre-incubation of HSV-1 with various concentrations of the 2kD peptide showed dose-dependent cytopathic effects (CPE) reduction patterns at concentrations from 6.25 to 50 microM. The presence of the 2kD peptide before HSV-1 infections showed moderate inhibition of virus-induced CPE as compared to during or after infections, with EC(50) values of 12.5, 6.25, and 6.25 microM, respectively. Similar results were observed when the 2kD peptide was assayed against bovine herpes virus (BHV), an enveloped virus like HSV-1. On the other hand, the 2kD peptide showed weak activity against poliovirus type 1, a non-enveloped virus. Taken together, these results indicate that the 2kD peptide was able not only to inhibit the initiation and the spread of infection, but also had an in vitro prophylactic effect against HSV-1 infection.

Megasome biogenesis in Leishmania amazonensis: a morphometric and cytochemical study.

Megasomes are large lysosomes found in the amastigote stage of Leishmania species belonging to the mexicana complex. The biogenesis of megasomes was investigated by transmission electron microscopy during the transformation of promastigotes into the amastigote form of L. amazonensis maintained in axenic cultures. Mainly small vacuoles with low electron density were found in the promastigote and early intermediate forms. Morphometrical analysis showed an increase in the volume density of these structures during the transformation process. Cysteine proteinase was localized in this structure by immunocytochemical assay. Membrane-bounded structures filled with electron-dense material were also found in significant amounts from the 2nd day on. These structures were relatively abundant, both in axenic and lesion-derived amastigotes, but not in stable long-term axenic amastigote culture. A three-dimensional reconstruction of lesion-derived amastigotes and axenic amastigotes of L. amazonensis demonstrated that megasomes comprise almost 5% of the total cell volume. In addition, the development of other organelles was examined during the transformation process.

Antibacterial activity of Ocimum gratissimum L. essential oil.

The essential oil (EO) of Ocimum gratissimum inhibited Staphylococcus aureus at a concentration of 0.75 mg/ml. The minimal inhibitory concentrations (MICs) for Shigella flexineri, Salmonella enteritidis, Escherichia coli, Klebsiella sp., and Proteus mirabilis were at concentrations ranging from 3 to 12 microg/ml. The endpoint was not reached for Pseudomonas aeruginosa (>=24 mg/ml). The MICs of the reference drugs used in this study were similar to those presented in other reports. The minimum bactericidal concentration of EO was within a twofold dilution of the MIC for this organism. The compound that showed antibacterial activity in the EO of O. gratissimum was identified as eugenol and structural findings were further supported by gas chromatography/mass spectra retention time data. The structure was supported by spectroscopic methods.

Fatty acid and sterol composition of three phytomonas species.

Fatty acid and sterol analysis were performed on Phytomonas serpens and Phytomonas sp. grown in chemically defined and complex medium, and P. françai cultivated in complex medium. The three species of the genus Phytomonas had qualitatively identical fatty acid patterns. Oleic, linoleic, and linolenic were the major unsaturated fatty acids. Miristic and stearic were the major saturated fatty acids. Ergosterol was the only sterol isolated from Phytmonas sp. and P. serpens grown in a sterol-free medium, indicating that it was synthesized de novo. When P. françai that does not grow in defined medium was cultivated in a complex medium, cholesterol was the only sterol detected. The fatty acids and sterol isolated from Phytomonas sp. and P. serpens grown in a chemically defined lipid-free medium indicated that they were able to biosynthesize fatty acids and ergosterol from acetate or from acetate precursors such as glucose or threonine.