The utility of banana peel extract agar in the presumptive identification of Cryptococcus neoformans.

We prepared a newer growth medium, banana peel extract agar (BPEA) containing the extracts of chopped banana peels for the selective cultivation of Cryptococcus neoformans. Over the medium, the growth resulted in the development of light to the dark brown coloured colonies indicating the chromogenic potential of the BPEA. The organism grown over BPEA was subsequently confirmed as C. neoformans by phenotypic as well as by molecular method. This medium, being cost-effective, may be used in resource-poor settings of the developing or underdeveloped countries for selective isolation of C. neoformans.

Antifungal and anti-inflammatory potential of eschweilenol C-rich fraction derived from Terminalia fagifolia Mart.

ETHNOPHARMACOLOGICAL RELEVANCE: Folk knowledge transmitted between generations allows traditional populations to maintain the use of medicinal plants for the treatment of several diseases. In this context, the species Terminalia fagifolia Mart., native to Brazil, is used for the treatment of chronic and infectious diseases. Plants rich in secondary metabolites, such as this species and their derivatives, may represent therapeutic alternatives for the treatment of diseases that reduce the quality of life of people. AIM OF THE STUDY: The aim of this study was to evaluate the antifungal and anti-inflammatory potential of aqueous fraction from ethanolic extract of T. fagifolia, with in silico study of the major compound of the fraction. MATERIAL AND METHODS: The phytochemical study of the aqueous fraction was performed by HPLC, LC/MS and NMR. The antifungal activity was evaluated against yeasts, by determination of the minimum inhibitory concentration and minimum fungicidal concentration. The effect on Candida albicans was analyzed by AFM. The antibiofilm potential against biofilms of C. albicans was also tested. The anti-inflammatory potential of the aqueous fraction was evaluated in vivo by the carrageenan-induced paw edema and peritonitis. A microglial model of LPS-induced neuroinflammation was also studied. Further insights on the activation mechanism were studied using quantum chemistry computer simulations. Toxicity was evaluated in the Galleria mellonella and human erythrocytes models. RESULTS: Eschweilenol C was identified as the major constituent of the aqueous fraction of the ethanolic extract of T. fagifolia. The aqueous fraction was active against all Candida strains used (sensitive and resistant to Fluconazole) with MICs ranging from 1000 to 0.4 µg/mL. By AFM it was possible to observe morphological alterations in treated Candida cells. The fraction significantly (p < 0.05) inhibited paw edema and decreased levels of malondialdehyde induced by carrageenan. In a microglial cell model, aqueous fraction demonstrated the ability to inhibit NF-κB after induction with lipopolysaccharide. The theoretical studies showed structural similarity between eschweilenol C and indomethacin and an excellent antioxidant potential. The aqueous fraction did not present toxicity in the studied models. CONCLUSION: The results indicate that the aqueous fraction of T. fagifolia has potential for biomedical applications with low toxicity. This finding can be attributed to the predominance of eschweilenol C in the aqueous fraction.

An ethnobotanical survey and antifungal activity of Piper guineense used for the treatment of fungal infections in West-African traditional medicine.

ETHNOBOTANICAL RELEVANCE: Piper guineense occurs commonly in West Africa where it is used for fungal infections instead of the costly and not always accessible conventional antifungals. Fungal, yeast-based diseases are common in West-Africa especially among those living with HIV/AIDS, and thus this study was performed in Imo state, South-Eastern Nigeria, where P. guineense is predominantly used for the treatment of fungal diseases, such as skin rashes, oral thrush and vaginosis. AIM OF STUDY: The scarce number of previous studies on the documentation of the traditional uses of P. guineense extracts for the treatment of fungal infections in Nigeria prompted this survey. The investigation focused on how traditional healers recognize and diagnose fungal infections, how P. guineense is collected, on the various parts used for the treatments, methods of preparations, administrations and treatments. In addition, an in vitro antifungal screening of P. guineense fruit and leaf extracts of various polarities, and piperine and piperlongumine, representing the main constituents in these extracts, were performed. METHODS: A house to house ethnobotanical survey was conducted using questionnaires. Twenty traditional medical practitioners (TMP) and herb sellers from ten villages were interviewed. Four human pathogenic strains of yeast and Cryptococcus neoformans, a yeast-like basidiomycete causing meningitis in immunocompromised individuals, were used for the antifungal screening. RESULTS: The traditional medical practitioners (TMP) and herb sellers explained that the leaves and fruits are the most commonly used plant parts for the treatments. The oral intake of the extracts in locally produced bamboo alcohol (Kai-kai) is the most common method of administration. In accordance with these recorded traditional uses, we found that extracts of P. guineense were growth inhibitory against the fungal strains with MIC values ranging from 39 to 2500 µg/mL. The lowest MIC value of 39 µg/mL was recorded for a methanol fruit extract against Candida albicans, C. glabrata and C. tropicalis. In addition, ethanol and hexane fruit extracts were effective against the growth of C. albicans and C. glabrata, respectively, with a MIC of 78 µg/mL. Piperlongumine and piperine were active against C. albicans with MIC values of 39 and 78 µg/mL respectively. CONCLUSION: P. guineense fruit and leaf extracts, as well as their piperamide alkaloid constituents piperine and piperlongumine, have interesting antifungal properties and could have potential as new antifungal scaffolds. Our results warrant further in-depth investigations to isolate and characterize piperamide alkaloids and other compounds responsible for the antifungal activity in the extracts.

Additive potential of combination therapy against cryptococcosis employing a novel amphotericin B and fluconazole loaded dual delivery system.

Cryptococcus neoformans is one of the most lethal fungi causing mortality across the globe. Immuno-competent patients and patients taking immuno-suppressive medications are extremely susceptible to its infection. For effective removal of cryptococcal burden, there is an urgent need for new forms of therapy. In the present study, we have explored the potential effects of amphotericin B (AMB) and fluconazole (FLC) in combination, against cryptococcosis in Swiss albino mice. To enhance the therapeutic potential of the tested drugs, they were entrapped into fibrin microspheres; a dual delivery vehicle comprising of poly-lactide co-glycolide (PLGA) microsphere that was additionally encapsulated into the fibrin cross-linked plasma bead. Dynamics of fibrin microspheres included survival and fungal burden in lung, liver and spleen of treated mice. While each drug was effective in combination or alone, prominent additive potential of AMB and FLC were clearly observed when used in fibrin microsphere. Significant reduction in fungal burden and increase in survival rate of AMB + FLC-fibrin microspheres treated mice shows an extensive accessibility of both tested drugs without any side-effects. A full potential of two-drug combination encapsulated in fibrin microspheres proposes an effective approach of safe delivery to the target site in their intact form and decrease the drug associated toxicities.

Anti-cryptococcal activity of ethanol crude extract and hexane fraction from Ocimum basilicum var. Maria bonita: mechanisms of action and synergism with amphotericin B and Ocimum basilicum essential oil.

CONTEXT: Ocimum basilicum L. (Lamiaceae) has been used in folk medicine to treat headaches, kidney disorders, and intestinal worms. OBJECTIVE: This study evaluates the anti-cryptococcal activity of ethanol crude extract and hexane fraction obtained from O. basilicum var. Maria Bonita leaves. MATERIALS AND METHODS: The MIC values for Cryptococcus sp. were obtained according to Clinical and Laboratory Standards Institute in a range of 0.3-2500 µg/mL. The checkerboard assay evaluated the association of the substances tested (in a range of 0.099-2500 µg/mL) with amphotericin B and O. basilicum essential oil for 48 h. The ethanol extract, hexane fraction and associations in a range of 0.3-2500 µg/mL were tested for pigmentation inhibition after 7 days of treatment. The inhibition of ergosterol synthesis and reduction of capsule size were evaluated after the treatment with ethanol extract (312 µg/mL), hexane fraction (78 µg/mL) and the combinations of essential oil + ethanol extract (78 µg/mL + 19.5 µg/mL, respectively) and essential oil + hexane fraction (39.36 µg/mL + 10 µg/mL, respectively) for 24 and 48 h, respectively. RESULTS: The hexane fraction presented better results than the ethanol extract, with a low MIC (156 µg/mL against C. neoformans T444 and 312 µg/mL against C. neoformans H99 serotype A and C. gattii WM779 serotype C). The combination of the ethanol extract and hexane fraction with amphotericin B and essential oil enhanced their antifungal activity, reducing the concentration of each substance needed to kill 100% of the inoculum. The substances tested were able to reduce the pigmentation, capsule size and ergosterol synthesis, which suggest they have important mechanisms of action. CONCLUSIONS: These results provide further support for the use of ethanol extracts of O. basilicum as a potential source of antifungal agents.

Recent advances in AIDS-related cryptococcal meningitis treatment with an emphasis on resource limited settings.

INTRODUCTION: Recent advances in the treatment and prevention of cryptococcal meningitis have the potential to decrease AIDS-related deaths. Areas covered: Targeted screening for asymptomatic cryptococcal antigenemia in persons with AIDS is a cost effective method for reducing early mortality in patients on antiretroviral therapy. For persons with symptomatic cryptococcal meningitis, optimal initial management with amphotericin and flucytosine improves survival compared to alternative therapies; however, amphotsericin is difficult to administer and flucytosine has not been available in middle or low income countries, where cryptococcal meningitis is most prevalent. Expert commentary: Improved care for cryptococcal meningitis patients in resource-limited settings is possible, and new treatment possibilities are emerging.

Quercetin and rutin as potential agents antifungal against Cryptococcus spp / Quercetina e rutina: potenciais agentes para terapia antifúngica

Abstract Amphotericin B is a fungicidal substance that is treatment of choice for most systemic fungal infections affecting as cryptococcosis the immunocompromised patients. However, severe side effects have limited the utility of this drug. The aim of this study was to evaluate the antifungal effect of the combination of amphotericin B with quercetin or rutin and as a protective of citotoxic effect. The antifungal activity to amphotericin B, quercetin and rutin alone and in combination was determined in Candida sp and Cryptococcus neoformans strains. Cytotoxicity test on erythrocytes was performed by spectrophotometric absorbance of hemoglobin. The amphotericin B MIC was reduced when used in combination with quercetin or rutin to C. neoformans ATCC strain and reduced when combined with rutin to a clinical isolate of C. neoformans. In addition, the combination of quercetin with amphotericin B may reduce the toxicity of amphotericin B to red blood cells. Our results suggest that quercetin and rutin are potential agents to combine with amphotericin B in order to reduce the amphotericin dose to lessen side effects and improve antifungal efficacy.

Resumo A anfotericina B é uma substância fungicida e é o tratamento de escolha para a maioria das infecções fúngicas sistêmicas que afetam os pacientes imunocomprometidos, como a criptococose. No entanto, as severas reações adversas têm limitado a utilização desta droga. O objetivo deste estudo foi avaliar o efeito antifúngico e o potencial efeito protetor de citotoxicidade da combinação de anfotericina B com quercetina ou rutina. A atividade antifúngica de anfotericina B, quercetina e rutina, isoladamente e em combinação foi determinada em cepas de Candida sp e Cryptococcus neoformans. O teste de citotoxicidade em eritrócitos foi realizado por espectrofotometria, através da determinação da absorbância da hemoglobina. A concentração inibitória mínima da anfotericina B foi reduzida quando utilizada em combinação com a quercetina e rutina em C. neoformans ATCC e reduzida quando combinados com rutina em um isolado clínico de C. neoformans. Além disso, a combinação de quercetina com anfotericina B pode reduzir a toxicidade da droga em eritrócitos. Os resultados sugerem que quercetina e rutina são potenciais agentes para combinação com anfotericina B, a fim de reduzir a dose de anfotericina, diminuindo os efeitos colaterais e melhorando sua eficácia antifúngica.

Chemical composition and biological activities of Artemisia judaica essential oil from southern desert of Jordan.

ETHNOPHARMACOLOGIC RELEVANCE: Artemisia judaica L. (Arabic name: Beithran), is a medicinal and aromatic plant growing in the valley bottoms of desert areas, particularly in the southern desert of Jordan nearest to the Jordan-Saudi Arabia borders and in Wadi Araba in the Southern Badia. In Jordan, A. judaica is widely used in traditional medicine being recommended by aboriginal Bedouins in the North Badia region of Jordan as calmative. Furthermore, it is used for the treatment of stomach ache, heart diseases, sexual weakness, diabetes, gastro-intestinal disorders and external wounding. Additionally, other folk medicines of the Arabic region commonly use this aromatic plant for the treatment of inflammatory-related diseases, for instance fungal infections, diabetes, atherosclerosis, cancer and arthritis. AIM OF THE STUDY: Considering the traditional medicinal uses and the lack of scientific studies addressing the cellular and molecular mechanisms behind A. judaica claimed activities, the present study was designed to validate some of the traditional uses ascribed to this species, specifically the antifungal and anti-inflammatory activities of A. judaica essential oil at doses devoid of cytotoxicity to mammalian cells. MATERIALS AND METHODS: Chemical analysis of A. judaica essential oil isolated by hydrodistillation from aerial parts was carried out by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The antifungal activity (minimal inhibitory concentrations and minimal lethal concentrations) was evaluated against yeasts, dermatophyte and Aspergillus strains. In order to deeply explore the mechanisms behind the anti-fungal effect of the essential oil, the germ tube inhibition assay and the biofilms formation assay were evaluated using Candida albicans. The assessment of cell viability was accomplished using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in both hepatocytes and macrophages. Furthermore, the in vitro anti-inflammatory potential of A. judaica oil was evaluated by measuring nitric oxide (NO) production using lipopolysaccharide (LPS)-stimulated mouse macrophages. RESULTS: Oxygen containing monoterpenes are a representative group of constituents (68.7%) with piperitone (30.4%), camphor (16.1%) and ethyl cinnamate (11.0%) as main compounds. The highest antifungal activity of the oil was observed against Cryptococcus neoformans, with a MIC value of 0.16µL/mL. The oil revealed an important inhibitory effect on germ tube formation in C. albicans with 80% inhibition of filamentation at a concentration of 0.16µL/mL. Importantly, the oil also interfered with pre-formed biofilms by reducing the amount of the attached biomass. Furthermore, the essential oil significantly inhibited NO production evoked by LPS on macrophages at concentrations with very low toxicity (0.32µL/mL) or without toxicity (0.16µL/mL) to both macrophages and hepatocytes. CONCLUSIONS: The present study revealed that A. judaica essential oil from Jordan significantly inhibited germ tube formation and disrupted preformed biofilms of C. albicans, emphasizing the therapeutic potential for the treatment of disseminated candidiasis. Additionally, safe concentrations of this essential oil significantly inhibited NO production elicited by LPS in macrophages, highlighting its potential anti-inflammatory activity. Overall, A. judaica bears promising therapeutic potential for further drug development. Importantly, this work also validates some of the traditional uses of A. judaica.

Toxicity of Amphotericin B Deoxycholate-Based Induction Therapy in Patients with HIV-Associated Cryptococcal Meningitis.

Amphotericin B deoxycholate (AmBd) is the recommended induction treatment for HIV-associated cryptococcal meningitis (CM). Its use is hampered by toxicities that include electrolyte abnormalities, nephrotoxicity, and anemia. Protocols to minimize toxicity are applied inconsistently. In a clinical trial cohort of AmBd-based CM induction treatment, a standardized protocol of preemptive hydration and electrolyte supplementation was applied. Changes in blood counts, electrolyte levels, and creatinine levels over 14 days were analyzed in relation to the AmBd dose, treatment duration (short course of 5 to 7 days or standard course of 14 days), addition of flucytosine (5FC), and outcome. In the 368 patients studied, the hemoglobin levels dropped by a mean of 1.5 g/dl (95% confidence interval [CI], 1.0 to 1.9 g/dl) following 7 days of AmBd and by a mean of 2.3 g/dl (95% CI, 1.1 to 3.6 g/dl) after 14 days. Serum creatinine levels increased by 37 µmol/liter (95% CI, 30 to 45 µmol/liter) by day 7 and by 49 µmol/liter (95% CI, 35 to 64µmol/liter) by day 14 of AmBd treatment. Overall, 33% of patients developed grade III/IV anemia, 5.6% developed grade III hypokalemia, 9.5% had creatinine levels that exceeded 220 µmol, and 6% discontinued AmBd prematurely. The addition of 5FC was associated with a slight increase in anemia but not neutropenia. Laboratory abnormalities stabilized or reversed during the second week in patients on short-course induction. Grade III/IV anemia (adjusted odds ratio [aOR], 2.2; 95% CI, 1.1 to 4.3; P = 0.028) and nephrotoxicity (aOR, 4.5; 95% CI, 1.8 to 11; P = 0.001) were risk factors for 10-week mortality. In summary, routine intravenous saline hydration and preemptive electrolyte replacement during AmBd-based induction regimens for HIV-associated CM minimized the incidence of hypokalemia and nephrotoxicity. Anemia remained a concerning adverse effect. The addition of flucytosine was not associated with increased neutropenia. Shorter AmBd courses were less toxic, with rapid reversibility.

Cyclopalladated Compound 7a Induces Apoptosis- and Autophagy-Like Mechanisms in Paracoccidioides and Is a Candidate for Paracoccidioidomycosis Treatment.

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 µg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses.

Increased Antifungal Drug Resistance in Clinical Isolates of Cryptococcus neoformans in Uganda.

Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/µl, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC50 of 4 µg/ml and an MIC90 of 8 µg/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC50 to 8 µg/ml and an MIC90 value of 32 µg/ml, with 31% of isolates with a fluconazole MIC of ≥ 16 µg/ml. We observed an amphotericin B MIC50 of 0.5 µg/ml and an MIC90 of 1 µg/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzed Cryptococcus susceptibility to sertraline, with an MIC50 of 4 µg/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P = 0.52).

Distinct and redundant roles of protein tyrosine phosphatases Ptp1 and Ptp2 in governing the differentiation and pathogenicity of Cryptococcus neoformans.

Protein tyrosine phosphatases (PTPs) serve as key negative-feedback regulators of mitogen-activated protein kinase (MAPK) signaling cascades. However, their roles and regulatory mechanisms in human fungal pathogens remain elusive. In this study, we characterized the functions of two PTPs, Ptp1 and Ptp2, in Cryptococcus neoformans, which causes fatal meningoencephalitis. PTP1 and PTP2 were found to be stress-inducible genes, which were controlled by the MAPK Hog1 and the transcription factor Atf1. Ptp2 suppressed the hyperphosphorylation of Hog1 and was involved in mediating vegetative growth, sexual differentiation, stress responses, antifungal drug resistance, and virulence factor regulation through the negative-feedback loop of the HOG pathway. In contrast, Ptp1 was not essential for Hog1 regulation, despite its Hog1-dependent induction. However, in the absence of Ptp2, Ptp1 served as a complementary PTP to control some stress responses. In differentiation, Ptp1 acted as a negative regulator, but in a Hog1- and Cpk1-independent manner. Additionally, Ptp1 and Ptp2 localized to the cytosol but were enriched in the nucleus during the stress response, affecting the transient nuclear localization of Hog1. Finally, Ptp1 and Ptp2 played minor and major roles, respectively, in the virulence of C. neoformans. Taken together, our data suggested that PTPs could be exploited as novel antifungal targets.

New acridone from the wood of Citrus reticulata Blanco.

A new acridone, named citruscridone (1) together with five known compounds were isolated from the wood of Citrus reticulata Blanco. Their structures were established based on spectroscopic evidence. The antibacterial and antifungal activities of the wood extracts and pure compounds were evaluated.

The copper interference with the melanogenesis OF Cryptococcus neoformans.

Melanin is a pigment produced by laccase, a phenoloxydase enzyme, and is related to the virulence of Cryptococcus neoformans as it is also considered an adaption mechanism to environmental conditions and protection against UV radiation, phagocytic system attack and antifungal drugs. Laccase synthesis is stimulated by several factors, including copper metabolism. The current study shows C. neoformans strains with higher melanization intensity when grown in L-dopa medium supplemented with different concentrations of copper sulfate. This increase shows that melanization rates may be enhanced in the presence of copper ions and may also enhance the virulence of C. neoformans in infected patients that present increasing copper concentrations in serum, such as those with HIV. The virulence of these strains may also be increased in the environment, where this metal is available as CuSO4 in algicidal and fungicidal compounds.

THE COPPER INTERFERENCE WITH THE MELANOGENESIS OF Cryptococcus neoformans / A interferência de cobre na melanogênese de Cryptococcus neoformans

Melanin is a pigment produced by laccase, a phenoloxydase enzyme, and is related to the virulence of Cryptococcus neoformans as it is also considered an adaption mechanism to environmental conditions and protection against UV radiation, phagocytic system attack and antifungal drugs. Laccase synthesis is stimulated by several factors, including copper metabolism. The current study shows C. neoformans strains with higher melanization intensity when grown in L-dopa medium supplemented with different concentrations of copper sulfate. This increase shows that melanization rates may be enhanced in the presence of copper ions and may also enhance the virulence of C. neoformans in infected patients that present increasing copper concentrations in serum, such as those with HIV. The virulence of these strains may also be increased in the environment, where this metal is available as CuSO4 in algicidal and fungicidal compounds.

A melanina é um pigmento produzido pela enzima lacase, uma fenoloxidase, e está associada à virulência de Cryptococcus neoformans sendo considerada mecanismo de adaptação às condições ambientais e proteção contra a radiação UV, ataque do sistema fagocítico e antifúngicos. A lacase tem sua síntese estimulada por diversos fatores, incluindo o metabolismo de cobre. Este estudo mostra linhagens de C. neoformans com maior intensidade de melanização quando cultivadas em meio L-dopa suplementado com diferentes concentrações de sulfato de cobre. Este aumento demonstra que as taxas de melanização podem ser aumentadas na presença de íons cobre e também aumentar a virulência de C. neoformans em pacientes infectados que apresentam aumento nas concentrações séricas de íons cobre tais como pacientes com HIV. A virulência destas linhagens também pode ser incrementada no meio ambiente, onde este metal está disponível como CuSO4 em compostos algicidas e fungicidas.

A survey of the in vitro antifungal activity of heather (Erica sp.) organic honey.

Monofloral heather (Erica sp.) honey samples (n=89), harvested in Portugal according to European organic beekeeping rules, were analyzed to test their antifungal effect against Candida albicans, Candida krusei, and Cryptococcus neoformans. A synthetic honey solution was also tested to determine antifungal activity attributable to sugars. The specific growth rate (µ) values showed that growth of all the yeasts was reduced in the presence of honey. The honey concentration (% wt/vol) that inhibited 10% of the yeast growth (X(min)) was 13.5% for C. albicans, 20.5% for C. krusei, and 17.1% for C. neoformans. The respective concentrations of heather honey and synthetic honey in the C. krusei culture medium above 60% (wt/vol) that inhibited 90% of the yeast growth (X(max)) and X(min), respectively, were established, whereas C. albicans and C. neoformans were more resistant because X(max) values were not reached over the range tested (10-60%, wt/vol). Heather honey might be tapped as a natural resource to look for new medicines for the treatment of mycotic infections. Further studies are now required to demonstrate if this antifungal activity has any clinical application.

Chemical composition and antifungal activity of the essential oils of Lavandula viridis L'Her.

In the present work we report for what we believe to be the first time the antifungal activity and mechanism of action of the essential oils of Lavandula viridis from Portugal. The essential oils were isolated by hydrodistillation and analysed by GC and GC/MS. The MIC and the minimal lethal concentration (MLC) of the essential oil and its major compounds were determined against several pathogenic fungi. The influence of subinhibitory concentrations of the essential oil on the dimorphic transition in Candida albicans was also studied, as well as propidium iodide and FUN-1 staining of Candida albicans cells by flow cytometry following short treatments with the essential oil. The oils were characterized by a high content of oxygen-containing monoterpenes, with 1,8-cineole being the main constituent. Monoterpene hydrocarbons were present at lower concentrations. According to the determined MIC and MLC values, the dermatophytes and Cryptococcus neoformans were the most sensitive fungi (MIC and MLC values ranging from 0.32 to 0.64 µl ml⁻¹), followed by Candida species (at 0.64-2.5 µl ml⁻¹). For most of these strains, MICs were equivalent to MLCs, indicating a fungicidal effect of the essential oil. The oil was further shown to completely inhibit filamentation in Candida albicans at concentrations well below the respective MICs (as low as MIC/16). Flow cytometry results suggested a mechanism of action ultimately leading to cytoplasmic membrane disruption and cell death. Our results show that L. viridis essential oils may be useful in the clinical treatment of fungal diseases, particularly dermatophytosis and candidosis, although clinical trials are required to evaluate the practical relevance of our in vitro research.

Perilla frutescens seed agar, a new medium for identification of the Cryptococcus species complex: evaluation for all major molecular types.

Here we present a new and simple medium made by Perilla frutescens seed as a tool for identification of the Cryptococcus species complex, namely Cryptococcus neoformans and Cryptococcus gattii. Its usefulness was evaluated for all major molecular types within the Cryptococcus species complex. As a result, this medium is better for identification of the species complex compared with Guizotia abyssinica or Helianthus annuus seed medium.

Antimicrobial activity of the extract and isolated compounds from Baccharis dracunculifolia D. C. (Asteraceae).

Baccharis dracunculifolia D.C. (Asteraceae) is the most important plant source of the Brazilian green propolis. Since propolis is known for its antimicrobial activity, the aim of this work was to evaluate the antimicrobial activities of B. dracunculifolia and some of its isolated compounds. The results showed that the leaves extract of B. dracunculifolia (BdE) presents antifungal and antibacterial activities, especially against Candida krusei and Cryptococcus neoformans, for which the BdE showed IC50 values of 65 microg mL(-1) and 40 microg mL(-1), respectively. In comparison to the BdE, it was observed that the green propolis extract (GPE) showed better antimicrobial activity, displaying an IC50 value of 9 microg mL(-1) against C. krusei. Also, a phytochemical study of the BdE was carried out, affording the isolation of ursolic acid (1), 2a-hydroxy-ursolic acid (2), isosakuranetin (3), aromadendrin-4'-methylether (4), baccharin (5), viscidone (6), hautriwaic acid lactone (7), and the clerodane diterpene 8. This is the first time that the presence of compounds 1, 2, and 8 in B. dracunculifolia has been reported. Among the isolated compounds, 1 and 2 showed antibacterial activity against methicillin-resistant Staphylococcus aureus, displaying IC50 values of 5 microg mL(-1) and 3 microg mL(-1), respectively. 3 was active against C. neoformans, showing an IC50 value of 15 microg mL(-1) and a MIC value of 40 microg mL(-1), while compounds 4-8 were inactive against all tested microorganisms. The results showed that the BdE, similar to the GPE, displays antimicrobial activity, which may be related to the effect of several compounds present in the crude extract.

Inhibition of chitin synthases and antifungal activities by 2'-benzoyloxycinnamaldehyde from Pleuropterus ciliinervis and its derivatives.

In the course of search for potent chitin synthase inhibitors from natural resources, a novel chitin synthases inhibitor, 2'-benzoyloxycinnamaldehyde (2'-BCA) (I), was isolated from the aerial parts of Pleuropterus ciliinervis NAKAI. 2'-BCA inhibited chitin synthase 1 and 2 of Saccharomyces cerevisiae with the IC50s of 54.9 and 70.8 microg/ml, respectively, whereas it exhibited no inhibitory activity for chitin synthase 3 up to 280 microg/ml. Its derivatives, 2'-chloro- (V) and 2(-bromo-cinnamaldehyde (VI), each showed 1.9 and 2.7-fold stronger inhibitory activities than 2'-BCA, with the IC50s of 37.2 and 26.6 microg/ml, respectively. Especially, the IC50 of compound VI against chitin synthase 2 represented 1.7-fold more potent inhibitory activity than polyoxin D, a well-known chitin synthase inhibitor. Furthermore, compounds V and VI showed potent antifungal activities against various fungi including human pathogenic fungi, with a particularly strong inhibitory activity against Cryptococcus neoformans (MIC = 16 microg/ml). Although the chemical synthesis of this compound has been reported, the present study is the first report to describe the isolation of 2'-BCA from natural resources and chitin synthases inhibitory activities of its derivatives. These results suggested that 2'-BCA and its derivatives can potentially serve as useful lead compounds for development of antifungal agents.