Self-assembling cashew gum-graft-polylactide copolymer nanoparticles as a potential amphotericin B delivery matrix.

Amphotericin B is an antibiotic used in the treatment of fungal disease and leishmania; however, it exhibits side effects to patients, hindering its wider application. Therefore, nanocarriers have been investigated as delivery systems for amphotericin B (AMB) in order to decrease its toxicity, besides increase bioavailability and solubility. Amphiphilic copolymers are interesting materials to encapsulate hydrophobic drugs such as AMB, hence copolymers of cashew gum (CG) and l-lactide (LA) were synthesized using two different CG:LA molar ratios (1:1 and 1:10). Data obtained revealed that copolymer nanoparticles present similar figures for particle sizes and zeta potentials; however, particle size of encapsulated AMB increases if compared to unloaded nanoparticles. The 1:10 nanoparticle sample has better stability although higher polydispersity index (PDI) if compared to 1:1 sample. High amphotericin (AMB) encapsulation efficiencies and low hemolysis were obtained. AMB loaded copolymers show lower aggregation pattern than commercial AMB solution. AMB loaded nanoparticles show antifungal activities against four C. albicans strains. It can be inferred that cashew gum/polylactide copolymers have potential as nanocarrier systems for AMB.

Evaluation of the antimicrobial and antioxidant activity of 7-hydroxy-4', 6-dimethoxy-isoflavone and essential oil from Myroxylon peruiferum L.f.

This study evaluated the antibacterial, antifungal, and antioxidant effect of 7-hydroxy-4',6-dimethoxy-isoflavone and essential oil of Myroxylon peruiferum. The compound was isolated and its structure elucidated by NMR. The chemical composition of essential oil determined by GC-MS analysis. To evaluation of antimicrobial activity, the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC) were performed. In addition to analysis of antioxidant activity, DPPH radical scavenging tests, iron chelating assay (FIC), antioxidant reducing power assay (FRAP) and ß-carotene bleaching assay (BCB) were performed. For the essential oil were identified 24 organized compounds having as main constituents; Germacrene D (17.2%), α-pinene (14.8%) and E-caryophyllene (10.8%). The results showed that isoflavone (2000 to 156 µg/mL) and essential oil (5.0 to 1.25%) present antibacterial and antifungal activity against Gram-positive bacteria and filamentous fungi. The isoflavone and the essential oil also presented antioxidant activity in all the tests, mainly on inhibition of the oxidation of ß-carotene test concentrations ranging from 60 to 100%. In conclusion, isoflavone and essential oil from M. peruiferum present an antimicrobial alternative against Gram-positive bacteria, especially of the genus Staphylococcus and dermatophyte fungi of the genus Trichophyton, as well as a natural compound antioxidant.

Insights into the candidacidal mechanism of Ctn[15-34] - a carboxyl-terminal, crotalicidin-derived peptide related to cathelicidins.

PURPOSE: Ctn[15-34], a carboxyl-terminal fragment of crotalicidin (a cathelicidin from the venom gland of a South American rattlesnake), has shown antifungal activity against clinical and standard strains of Candida species. The aim of the present work was to investigate the underlying mechanisms of the candidicidal activity of Ctn[15-34]. METHODOLOGY: The time-kill profile and drug synergism were evaluated by means of a microdilution assay and multi-parametric flow cytometry. The presumptive interaction of Ctn[15-34] with lipid membranes was estimated in vitro with a lipid-mimic compound, the chromogenic substance 4-nitro-3-(octanoyloxy)benzoic acid (4N3OBA).Results/Key findings. The absorbance increment (at 425 nm) indicated a concentration- and time-dependent in-solution association between Ctn[15-34] and 4N3OBA. The interaction of Ctn[15-34] with Candida cells was confirmed by flow cytometric measurements with the 5(6)-carboxyfluorescein-labelled peptide (CF-Ctn[15-34]). Analysis of the killing time of Candida exposed to Ctn[15-34] and amphotericin B (AMB) showed that both the peptide and polyene drug reduce the number of c.f.u. but in mechanistically different ways. The Ctn[15-34] peptide alone caused yeast cell membrane disruption, which was confirmed by lactate dehydrogenase leakage and biomarkers of cell death mediated by necrosis. CONCLUSION: Overall, Ctn[15-34] displays a synergistic antifungal activity with AMB, an effect that can be further developed into a multi-target therapeutic option with other antimycotics currently in use.

Anacardic Acid Constituents from Cashew Nut Shell Liquid: NMR Characterization and the Effect of Unsaturation on Its Biological Activities.

Anacardic acids are the main constituents of natural cashew nut shell liquid (CNSL), obtained via the extraction of cashew shells with hexane at room temperature. This raw material presents high technological potential due to its various biological properties. The main components of CNSL are the anacardic acids, salicylic acid derivatives presenting a side chain of fifteen carbon atoms with different degrees of unsaturation (monoene-15:1, diene-15:2, and triene-15:3). Each constituent was isolated by column chromatography using silica gel impregnated with silver nitrate. The structures of the compounds were characterized by nuclear magnetic resonance through complete and unequivocal proton and carbon assignments. The effect of the side chain unsaturation was also evaluated in relation to antioxidant, antifungal and anticholinesterase activities, and toxicity against Artemia salina. The triene anacardic acid provided better results in antioxidant activity assessed by the inhibition of the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), higher cytotoxicity against A. salina, and acetylcholinesterase (AChE) inhibition. Thus, increasing the unsaturation of the side chain of anacardic acid increases its action against free radicals, AChE enzyme, and A. salina nauplii. In relation to antifungal activity, an inverse result was obtained, and the linearity of the molecule plays an important role, with monoene being the most active. In conclusion, the changes in structure of anacardic acids, which cause differences in polarity, contribute to the increase or decrease in the biological activity assessed.

Antifungal activity of plant extracts against microsporum canis and candida spp. strains / Actividad antifúngica de extractos de plantas contra cepas de microsporum canis y candida spp

Aiming to find a phytotherapeutic compounds to treat animal fungal infections, plants commonly found in Northeastern Brazil were evaluated in vitro against Microsporum canis and Candida spp. strains isolated from dogs and cats. The leaf ethanol extracts of Momordica charantia, Calotropis procera, Peschiera affinis and Piper tuberculatum and decoction of Mangifera indica were initially evaluated by the agar-well diffusion method. Four extracts induced growth inhibition zones against M. canis: P. tuberculatum (20 mm), M. indica (14 mm), M. charantia (13 mm) and P. affinis (11 mm). None of them were active against Candida spp. Broth microdilution tests were performed for M. canis strains (n=5), to find the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC). The geometric means for the MIC values were 590, 370, 350, 170 ug/mL, and for the MFC values were 1190, 750, 700, 340 ug/mL for M. charantia, P. affinis, P. tuberculatum and M. indica, respectively. Therefore, extracts from M. charantia, P. affinis, P. tuberculatum and M. indica are good candidates to produce antifungal phytotherapics since these extracts demonstrated good activity against M. canis.

Con el objetivo de encontrar compuestos fitoterapéuticos para tratar las infecciones por hongos de los animales, plantas que se encuentran comúnmente en el noreste de Brasil se evaluaron in vitro frente a cepas de Microsporum canis y Candida spp. aisladas de perros y gatos. Los extractos etanólicos de hojas de Momordica charantia, Calotropis procera, Peschiera affinis y Piper tuberculatum y la decocción de Mangifera índica fueron evaluados inicialmente por el método de difusión en pocillos de agar. Cuatro extractos indujeron zonas de inhibición del crecimiento contra M. canis: P. tuberculatum (20 mm), M. índica (14 mm), M. charantia (13 mm) y P. affinis (11 mm). Ninguno de ellos fue activo contra Candida spp. Se realizaron pruebas de microdilución en caldo para las cepas de M. canis (n = 5), para encontrar la concentración mínima inhibitoria (CIM) y la concentración fungicida mínima (CFM). Las medias geométricas de los valores de CIM fueron 590, 370, 350, 170 mg/ml, y para los valores de CFM fueron 1.190, 750, 700, 340 mg/ml de M. charantia, P. affinis, P. tuberculatum y M. indica, respectivamente. Por lo tanto, los extractos de M. charantia, P. affinis, P. tuberculatum y M. indica son buenos candidatos para la producción de fitoterápicos antifúngicos ya que estos extractos demostraron una buena actividad contra M. canis.

Alkylphenol Activity against Candida spp. and Microsporum canis: A Focus on the Antifungal Activity of Thymol, Eugenol and O-Methyl Derivatives.

In recent years there has been an increasing search for new antifungal compounds due to the side effects of conventional antifungal drugs and fungal resistance. The aims of this study were to test in vitro the activity of thymol, eugenol, estragole and anethole and some O-methyl-derivatives (methylthymol and methyleugenol) against Candida spp. and Microsporum canis. The broth microdilution method was used to determine the minimum inhibitory concentration (MIC). The minimum fungicidal concentrations (MFC) for both Candida spp. and M. canis were found by subculturing each fungal suspension on potato dextrose agar. Thymol, methylthymol, eugenol, methyl-eugenol, anethole, estragole and griseofulvin respectively, presented the following MIC values against M. canis: 4.8-9.7; 78-150; 39; 78-150; 78-150; 19-39 µg/mL and 0.006-2.5 mg/mL. The MFC values for all compounds ranged from 9.7 to 31 µg/mL. Concerning Candida spp, thymol, methylthymol, eugenol, methyleugenol, anethole, estragole and amphotericin, respectively, showed the following MIC values: 39; 620-1250; 150-620; 310-620; 620; 620-1250 and 0.25-2.0 mg/mL. The MFC values varied from 78 to 2500 µg/mL. All tested compounds thus showed in vitro antifungal activity against Candida spp. and M. canis. Therefore, further studies should be carried out to confirm the usefulness of these alkylphenols in vivo.

PCR-AGE, automated and manual methods to identify Candida strains from veterinary sources: a comparative approach.

The increasing incidence of candidiasis has drawn the attention of scientists and clinicians attempting to improve methods of studying Candida yeasts. PCR amplification followed by agarose gel electrophoresis (PCR-AGE) and the manual method (morphological characteristics, biochemical profiles and culturing on CHROMagar-Candida) and VITEK 2 automated method were used to test a total of 30 fungal strains from dog sources. The strains were obtained from cases of dermatitis, otitis externa and from the ears, oral mucosa, vaginal mucosa, prepuce and perianal region of clinically normal dogs. After identification as Candida yeasts by the manual method, the strains were analyzed using both VITEK and PCR-AGE methods. Isolates of C. parapsilosis ATCC 22019, C. krusei ATCC 6258 and C. albicans ATCC 10231 were included as controls. The universal primers ITS1, ITS3 and ITS4 were used in two independent PCR reactions. Of 30 yeast isolates, 3 isolates (Saccharomyces cerevisiae, C. rugosa and C. parapsilosis) that were incompletely identified by the manual method were identified with the PCR-AGE and VITEK methods. The results revealed a 96.7% and 86.7% concurrent identification between the PCR-AGE and VITEK methods versus the manual method, respectively. PCR-AGE showed a greater level of concordance with the manual method, besides being faster and more sensitive than the other methods examined, and is therefore indicated for routine diagnostic testing of Candida spp. strains from veterinary sources.

The anatomical distribution and antimicrobial susceptibility of yeast species isolated from healthy dogs.

The aim of this work was to identify the predominant yeast species present at different anatomical sites in healthy dogs and to determine their in vitro antimicrobial susceptibility using a broth microdilution assay. Samples were collected from the preputial, vaginal, oral and perianal mucosae and the isolates cultured were identified according to their morphological characteristics and biochemical profile. Malassezia pachydermatis was the most commonly isolated yeast, followed by Candida parapsilosis, Candida tropicalis, Candida albicans, Saccharomyces cerevisiae and Rhodotorula spp. Minimum inhibitory concentrations of the azole derivatives ketoconazole, itraconazole and fluconazole against Candida spp. were 0.03-16 microg/mL, 0.06 to >16 microg/mL and 0.5-64 microg/mL, respectively and Candida isolates were sensitive to caspofungin and amphotericin B. Although all isolates of M. pachydermatis were sensitive to itraconazole, fluconazole, ketoconazole and amphotericin B, they were found to be resistant to caspofungin. The study has highlighted that Candida spp., M. pachydermatis, S. cerevisiae and Rhodotorula spp. are part of the normal canine surface microbiota and some of these organisms exhibit in vitro resistance to commonly used antimicrobials.

Phenotypic characterization and in vitro antifungal sensitivity of Candida spp. and Malassezia pachydermatis strains from dogs.

Yeasts of the genera Candida and Malassezia can be found as commensal microorganisms in animals. The main species of importance in veterinary medicine are Malassezia pachydermatis and Candida albicans. The objectives of this study were to conduct a phenotypic characterization and to evaluate the in vitro antifungal sensitivity of strains of C. albicans (n=5), C. tropicalis (n=3) and M. pachydermatis (n=32) isolated from dogs. The phenotyping was based on macro and micromorphological features as well as biochemical analysis. The techniques of microdilution in broth and dilution in agar were used to evaluate the in vitro sensitivity of Candida spp. and M. pachydermatis, respectively. The tested drugs were ketoconazole (KTC), itraconazole (ITC), fluconazole (FLC) and amphotericin B (AMB). The morphological analysis of the strains of Candida spp. and M. pachydermatis did not show any noteworthy alterations when compared to standard strains. On the other hand, in the biochemical tests, 34.4% of the strains of M. pachydermatis were negative for the urease test. Four strains of C. albicans were resistant to FLC with a minimum inhibitory concentration (MIC) >64microg/mL and all were resistant to KTC and ITC (MIC>16microg/mL). The MIC for two strains of C. tropicalis were >16microg/mL for KTC and ITC, and >64microg/mL for FLC. It is worth highlighting that all of the strains tested were sensitive to AMB with the MIC varying from 0.25-1.0microg/mL. All strains of M. pachydermatis were sensitive to ITC with a minimum fungistatic concentration (MFC) 0.0075microg/mL. The MIC for 29 strains was the same (MFC0.0075microg/mL) for KTC. The MFCs for FLC varied from 1 to 16microg/mL, and for AMB, the MFC interval was 0.125-8microg/mL. There were no alterations in the classic phenotypic features of the strains of Candida spp. and M. pachydermatis isolated from dogs but, unlike M. pachydermatis, Candida spp. were much more resistant to azole antifungal agents.