Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin.

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 µg ml-1 while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.

Bovine serum albumin nanoparticles containing amphotericin B were effective in treating murine cutaneous leishmaniasis and reduced the drug toxicity.

Cutaneous leishmaniasis is the most common form of leishmaniasis and the available chemotherapy causes serious side effects, justifying the search for new therapies. This study investigated the antileishmanial activity of bovine serum albumin (BSA) nanoparticles containing amphotericin B (AmB) against Leishmania amazonensis. The antiproliferative activity against promastigotes and amastigotes was assessed and the cytotoxicity was determined and compared to commercial AmB-deoxycholate (AmB-D). In vivo antileishmania activity was evaluated in murine cutaneous leishmaniasis model. BSA nanoparticles showed spherical shape, mean size about 180 nm, zeta potential of ≈ -45 mV and AmB encapsulation efficiency >95%. AmB-D was effective in promastigote and amastigote forms, while AmB-loaded BSA nanoparticles were more effective against amastigotes than promastigotes. AmB-D was more effective than AmB-loaded BSA nanoparticles in both forms, however, the lowest cytotoxicity against macrophages was achieved by AmB-nanoparticles. BALB/c mice treated with AmB-D or AmB-loaded BSA nanoparticles showed a significant decrease in the lesion thickness at the infected footpad. Histopathological analysis after 3 weeks of treatment revealed AmB-D-related toxicity in heart, spleen, lung, liver and kidneys, while treatment with AmB-loaded BSA nanoparticles did not reveal tissue toxicity. The antileishmanial efficacy and the reduced toxicity become BSA nanoparticles containing AmB a potential candidate for treating cutaneous leishmaniasis.

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.

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.

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.

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.

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.

Chlorophyll treatment combined with photostimulation increases glycolysis and decreases oxidative stress in the liver of type 1 diabetic rats.

Photodynamic therapy (PDT) promotes cell death, and it has been successfully employed as a treatment resource for neuropathic complications of diabetes mellitus (T1DM) and hepatocellular carcinoma. The liver is the major organ involved in the regulation of energy homeostasis, and in pathological conditions such as T1DM, changes in liver metabolic pathways result in hyperglycemia, which is associated with multiple organic dysfunctions. In this context, it has been suggested that chlorophyll-a and its derivatives have anti-diabetic actions, such as reducing hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, but these effects have not yet been proven. Thus, the biological action of PDT with chlorophyll-a on hepatic parameters related to energy metabolism and oxidative stress in T1DM Wistar rats was investigated. Evaluation of the acute effects of this pigment was performed by incubation of isolated hepatocytes with chlorophyll-a and the chronic effects were evaluated by oral treatment with chlorophyll-based extract, with post-analysis of the intact liver by in situ perfusion. In both experimental protocols, chlorophyll-a decreased hepatic glucose release and glycogenolysis rate and stimulated the glycolytic pathway in DM/PDT. In addition, there was a reduction in hepatic oxidative stress, noticeable by decreased lipoperoxidation, reactive oxygen species, and carbonylated proteins in livers of chlorophyll-treated T1DM rats. These are indicators of the potential capacity of chlorophyll-a in improving the status of the diabetic liver.

Antibacterial activity of indole alkaloids from Aspidosperma ramiflorum.

We evaluated the antibacterial activities of the crude methanol extract, fractions (I-V) obtained after acid-base extraction and pure compounds from the stem bark of Aspidosperma ramiflorum. The minimum inhibitory concentration (MIC) was determined by the microdilution technique in Mueller-Hinton broth. Inoculates were prepared in this medium from 24-h broth cultures of bacteria (10(7) CFU/mL). Microtiter plates were incubated at 37 masculineC and the MICs were recorded after 24 h of incubation. Two susceptibility endpoints were recorded for each isolate. The crude methanol extract presented moderate activity against the Gram-positive bacteria B. subtilis (MIC = 250 microg/mL) and S. aureus (MIC = 500 microg/mL), and was inactive against the Gram-negative bacteria E. coli and P. aeruginosa (MIC > 1000 microg/mL). Fractions I and II were inactive against standard strains at concentrations of < or =1000 microg/mL and fraction III displayed moderate antibacterial activity against B. subtilis (MIC = 500 microg/mL) and S. aureus (MIC = 250 microg/mL). Fraction IV showed high activity against B. subtilis and S. aureus (MIC = 15.6 microg/mL) and moderate activity against E. coli and P. aeruginosa (MIC = 250 microg/mL). Fraction V presented high activity against B. subtilis (MIC = 15.6 microg/mL) and S. aureus (MIC = 31.3 microg/mL) and was inactive against Gram-negative bacteria (MIC > 1000 microg/mL). Fractions III, IV and V were then submitted to bioassay-guided fractionation by silica gel column chromatography, yielding individual purified ramiflorines A and B. Both ramiflorines showed significant activity against S. aureus (MIC = 25 microg/mL) and E. faecalis (MIC = 50 microg/mL), with EC50 of 8 and 2.5 microg/mL for ramiflorines A and B, respectively, against S. aureus. These results are promising, showing that these compounds are biologically active against Gram-positive bacteria.

Antimicrobial effects of Piper hispidum extract, fractions and chalcones against Candida albicans and Staphylococcus aureus.

Three chalcones, 2'-hydroxy-4,4',6'-trimethoxychalcone, 2'-hydroxy-4,4',6'-tetramethoxychalcone, and 3,2'-dihydroxy-4,4',6'-trimethoxychalcone, were isolated from the leaves of Piper hispidum in a bioguided fractionation of crude extract. The antimicrobial activity of crude extract of P. hispidum leaves was determined against bacteria Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and yeasts Candida albicans, C. parapsilosis and C. tropicalis. Fractions and chalcones were tested against C. albicans and S. aureus. The checkerboard assay was performed to assess synergic interactions between extract and antifungal drugs, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was used to evaluate anti-biofilm effects of extract. The extract was active against yeasts, S. aureus and B. subtilis with MIC values between 15.6 and 62.5µg/mL. Synergistic effects of extract associated with fluconazole and nystatin were observed against C. albicans, with fractional inhibitory concentration indices of 0.37 and 0.24, respectively. The extract was also effective against C. albicans and S. aureus biofilm cells at concentrations of 62.5 and 200µg/mL, respectively. Thus, P. hispidum may be a possible source of bioactive substances with antimicrobial properties.

Antiprotozoal and molluscicidal activities of five Brazilian plants.

Leishmaniasis, Chagas' disease and schistosomiasis (bilharzia) are parasitic diseases with wide distribution on the American continent, affecting millions of people. In the present study, biological assays for antiprotozoal and molluscicidal activities were carried out with ethanolic extracts of plant species from the Brazilian part of the Upper Paraná River. Crude extracts were obtained by percolation with absolute ethanol from the leaves of Cayaponia podantha Cogn., Nectandra falcifolia (Nees) Castiglioni and Paullinia elegans Cambess., as well as from the aerial parts of Helicteres gardneriana St. Hil. & Naud. and Melochia arenosa Benth., all belonging to genera used in folk medicine. Trypanocidal activity of plants was assayed on epimastigote cultures in liver infusion tryptose. Anti-leishmanial activity was determined over cultures of promastigote forms of the parasite in Schneider's Drosophila medium. Microscopic countings of parasites, after their incubation in the presence of different concentrations of the crude extracts, were made in order to determine the percentage of growth inhibition. C. podantha and M. arenosa, at a concentration of 10 microg/mL, showed 90.4 +/- 11.52 and 88.9 +/- 2.20% growth inhibition, respectively, of epimastigote forms of Trypanosoma cruzi, whereas N. falcifolia demonstrated an LD50 of 138.5 microg/mL against promastigote forms of Leishmania (Viannia) braziliensis. Regarding molluscicidal activity, the acute toxicity of the extracts on Biomphalaria glabrata was evaluated by a rapid screening procedure. M. arenosa was 100% lethal to snails at 200 microg/mL and showed an LD50 of 143 microg/mL. Screening of plant extracts represents a continuous effort to find new antiparasitic drugs.

Antidermatophytic activity of hydroalcoholic extracts from Rosmarinus officinalis and Tetradenia riparia.

Rosmarinus officinalis and Tetradenia riparia are used in folk medicine for the treatment of disease, including infectious diseases and skin disorders. The purpose of this study was to investigate the antifungal activity of hydroalcoholic extracts from R. officinalis and T. riparia against strains of Trichophyton rubrum, T. mentagrophytes and Microsporum gypseum. Hydroalcoholic extracts prepared with dried leaves from R. officinalis, Psidium guajava and T. riparia were assayed against dermatophyte species by the microdilution technique and by microscopy. R. officinalis and T. riparia were the most active against dermatophytes, as determined from the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC), and were investigated further. Fluorescence microscopy and scanning electron microscopy were used to investigate inhibition of hyphal growth by the two extracts, and showed a strong inhibition and an irregular growth pattern. Both extracts showed good action against dermatophytes, inhibiting fungal growth and causing alterations in their hyphae. Therefore, R. officinalis and T. riparia are potential sources of new compounds for the development of antifungal drugs.

Chlorophyll treatment combined with photostimulation increases glycolysis and decreases oxidative stress in the liver of type 1 diabetic rats

Photodynamic therapy (PDT) promotes cell death, and it has been successfully employed as a treatment resource for neuropathic complications of diabetes mellitus (T1DM) and hepatocellular carcinoma. The liver is the major organ involved in the regulation of energy homeostasis, and in pathological conditions such as T1DM, changes in liver metabolic pathways result in hyperglycemia, which is associated with multiple organic dysfunctions. In this context, it has been suggested that chlorophyll-a and its derivatives have anti-diabetic actions, such as reducing hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, but these effects have not yet been proven. Thus, the biological action of PDT with chlorophyll-a on hepatic parameters related to energy metabolism and oxidative stress in T1DM Wistar rats was investigated. Evaluation of the acute effects of this pigment was performed by incubation of isolated hepatocytes with chlorophyll-a and the chronic effects were evaluated by oral treatment with chlorophyll-based extract, with post-analysis of the intact liver by in situ perfusion. In both experimental protocols, chlorophyll-a decreased hepatic glucose release and glycogenolysis rate and stimulated the glycolytic pathway in DM/PDT. In addition, there was a reduction in hepatic oxidative stress, noticeable by decreased lipoperoxidation, reactive oxygen species, and carbonylated proteins in livers of chlorophyll-treated T1DM rats. These are indicators of the potential capacity of chlorophyll-a in improving the status of the diabetic liver.

Cloning and characterization of the metacyclogenin gene, which is specifically expressed during Trypanosoma cruzi metacyclogenesis.

We isolated a gene that is differentially expressed during Trypanosoma cruzi metacyclogenesis by the representation of differential expression (RDE) method, using differentiating epimastigotes cultured in chemically defined medium. This gene, the metacyclogenin gene, encodes a 630-nucleotide mRNA that is specifically associated with the polysomes of epimastigotes allowed to differentiate for 24 h. We sequenced and characterized the metacyclogenin gene and found that there were at least three copies of the gene organized into tandem 2.8 kb repeats in the genome of T. cruzi Dm28c. We analyzed the repeats and found that they contained two other genes, one encoding tryparedoxin peroxidase and the other encoding a 0.6 kb mRNA (named associated gene or AG) with sequences showing no significant similarity to those in the GenBank database. Northern blot analysis of polysomal RNA extracted from replicating and differentiating epimastigotes showed that metacyclogenin and AG genes displayed similar patterns of expression. Their products were detected only in differentiating epimastigotes, whereas tryparedoxin peroxidase was detected only in the polysomal RNA fraction of replicating and differentiating epimastigotes. In Northern blots of total RNA from differentiating and replicating epimastigotes, the genes studied were detected in both cell populations. The differential expression of the metacyclogenin gene was confirmed by immunocytochemistry studies showing that the protein is detected only in differentiating (adhered) epimastigote. The results suggest that mRNA mobilization to polysomes is an important mechanism in the regulation of gene expression in T. cruzi.

Evaluation of the antioedematogenic, free radical scavenging and antimicrobial activities of aerial parts of Tillandsiastreptocarpa Baker-Bromeliaceae.

The crude methanolic extract of the aerial parts of Tillandsiastreptocarpa was investigated for their acute toxicity and antioedematogenic, antioxidant and antimicrobial activities. Also, the antioedematogenic activity of the hexane fraction resulting from the partition of the crude methanolic extract was evaluated. The methanolic extract and the hexane fraction showed significant (P < 0.05) inhibition of ear oedema, observed at 2 mg/ear in the croton oil-induced mice ear oedema test. In the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging test, a high reactivity and potent antioxidant effect (IC(50) = 0.0056%, w/v) were observed for the methanolic extract. The antimicrobial activity assay showed that the crude methanolic extract was inactive toward Escherichiacoli, Staphylococcusaureus, Pseudomonasaeruginosa, Bacillussubtilis, Candidaalbicans, C. parapsilosis, C. krusei and C. tropicalis (MIC > 500 microg/ml). The methanolic extract showed no toxic effect on mice at a single dose of 2000 mg/kg (p.o). Common side effects including mild diarrhoea, loss of weight and depression were not recorded. The compounds cycloartenol, 4',5-dihydroxy-3',7-dimethoxyflavanone and a mixture of the steroids stigmasterol, beta-sitosterol and campesterol, were isolated from the hexane fraction and identified by spectroscopic methods.

Microbial populations of an upflow anaerobic sludge blanket reactor treating wastewater from a gelatin industry.

The microbial populations of an upflow anaerobic sludge blanket reactor, used for treating wastewater from the gelatin industry, were studied by microbiological methods and phase-contrast and electron microscopy. Microscopy examination of the sludge showed a complex mixture of various rod-shaped and coccoid bacterial pluslong filaments and verymobile curved rods. In addition free-living anaerobic ciliates and flagellates were also observed. The trophic group population observed in decreasing order of dominance were hydrolytic and acetogenic at 10(6) and sulfate reducing and methanogenic at 10(5). The rate of methane production in anaerobic granular sludge cultivated in growth medium supplement with formate pressurized with H2:CO2 showed a significant increase in methane yield compared with theseed culture containingthe same substrate and atmosphere of N2:CO2. Similar rates of methane production were observed when the growth medium was supplemented with acetate pressurized either with H2:CO2 or N2:CO2. The number of total anaerobic bacteria at 10(7), fecal coliforms and total coliforms at 10(6), and fecal streptococci at 10(3) is based on colony counts on solid media. The four prevalent species of facultative anaerobic gram-negative bacteria that belong to the family of Enterobacteriaceae were identified as Escherichia coli, Esherichia fergusonii, Klebsiella oxytoca, and Citrobacter freundii. The species Aeromonas hydrophila, Aeromonas veronii, Acinetobacter iwoffi and Stenotrophomonas maltophila were the most frequently isolated glucose fermenting and nonfermenting gram-negative bacilli.

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.

Antimicrobial activity of synthetic bornyl benzoates against Trypanosoma cruzi.

We report here for the first time the in vitro effects of (1S,2R,4S)-1,7,7-trimethyl-bicyclo[2·2·1]heptan-2-yl-3',4',5'-trimethoxy benzoate (1) and (1S,2R,4S)-1,7,7-trimethyl-bicyclo[2·2·1]heptan-2-yl benzoate (2) on the growth and ultrastructure of Trypanosoma cruzi. These two synthetic compounds exerted an antiproliferative effect on the epimastigote forms of the parasite. The ICs(50/72h) of two synthetic L-bornyl benzoates, 1 and 2, was 10·1 and 12·8 µg/ml, respectively. Both compounds were more selective against epimastigotes than HEp-2 cells. Ultrastructural analysis revealed intense cytoplasmic vacuolization and the appearance of cytoplasmic materials surrounded by membranes. The treatment of peritoneal macrophages with compounds 1 and 2 caused a significant decrease in the number of T. cruzi-infected cells. L-Bornyl benzoate derivatives may serve as a potential source for the development of more effective and safer chemotherapeutic agents against T. cruzi infections.

Effects of a methanolic fraction of soybean seeds on the transcriptional activity of peroxisome proliferator-activated receptors (PPAR).

Since the anti-inflammatory, antidiabetic and hypolipidemic effects of soy isoflavones may be mediated by activation of peroxisome proliferator-activated receptors (PPAR), the present study investigated whether the methanolic fractions obtained from soybean seeds (E1) and soybean seed coats with hypocotyls (E2) could influence PPARalpha, PPARgamma and PPARbeta/delta transcriptional activity. The isoflavones from E1 and E2 were quantified by HPLC analysis. E1 and E2 were rich in isoflavones (daidzin, glycitin, genistin, malonyldaidzin, malonylglycitin, malonylgenistin, daidzein, glycitein, and genistein). Moreover, E1 and E2 showed no evidence of genetically modified material containing the gene CP4 EPSPS. To investigate PPAR transcriptional activity, human promonocytic U-937 cells were treated with E1 and E2 (200, 400, 800, and 1600 microg/mL), positive controls or vehicle. Data are reported as fold-activation of the luciferase reporter driven by the PPAR-responsive element. Dose-response analysis revealed that E1 and E2 induced the transcriptional activity of PPARalpha (P < 0.001), with activation comparable to that obtained with 0.1 mM bezafibrate (positive control) at 1600 microg/mL (4-fold) and 800 microg/mL (9-fold), respectively. In addition, dose-response analysis revealed that E1 and E2 activated PPARbeta/delta (P < 0.05), and the activation at 800 microg/mL (4- and 9-fold, respectively) was comparable to that of 0.1 mM bezafibrate (positive control). However, no effect on PPARgamma was observed. Activation of PPARalpha is consistent with the lipid-lowering activity of soy isoflavones in vivo, but further studies are needed to determine the physiological significance of PPARbeta/delta activation.