Laura Svetaz

Value of the ethnomedical information for the discovery of plants with antifungal properties. A survey among seven Latin American countries.

ETHNOPHARMACOLOGICAL RELEVANCE This study reports the antifungal evaluation of 327 plant species (92 families and 251 genera) from seven Latin American countries which were selected on the basis of their reported ethnomedical uses and compared them with plants selected at random. AIM OF THE STUDY (a) The main aim of this study was to investigate whether the probability of detecting antifungal plants is higher when plants have reports of ethnopharmacological uses related to fungal infections (PAU group) than when they are selected at random (PNAU group). (b) The second objective was to determine, within the PAU group, whether the probability of obtaining a positive result will be higher when the plants are tested against dermatophytes, than against yeasts or Aspergillus spp. (c) The third goal was to investigate, within all MICs<or=1000 microg/mL, if the MICs displayed by the PAU group are comparatively lower than MIC values of the PNAU group; that is to say, if they can be expected more potent antifungal plants within the group of plants that have a history of traditional use related to fungal infections than when they do not have one. MATERIALS AND METHODS A five-stage process of documentation, evaluation and analysis of results was conducted: (1) selection of words that could describe the ethnopharmacological use related to fungal infections; (2) a survey of specialized literature in each country; (3) collection and preparation of an extract of each plant; (4) antifungal evaluation of the selected plants and (5) statistical analysis of the results. For the antifungal evaluation, the microbroth dilution assay recommended by the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) was used against a panel of eleven human opportunistic and pathogenic fungi. For the statistical analysis the Pearsons Chi Square test and the Scores test were used. RESULTS (a) A significantly higher probability of detecting plants with antifungal activity against at least one fungus was found within the PAU (40.3%) than the PNAU group (21.3%) (p<0.01). (b) A similar higher probability than in (a) (39.6% vs. 20.8%) was found when plants were tested against dermatophytes (p<0.01) but not against yeasts or Aspergillus spp. (p>0.05). (c) Within the detected antifungal plants from both groups, plants of the PAU group displayed higher activities (lower MICs) than those of PNAU group against dermatophytes (p<0.05) but not against yeasts or Aspergillus spp. CONCLUSIONS Considering that dermatophytes are the cause of superficial fungal infections, which can be easily detected and followed by traditional healers, our findings suggest that the ethnopharmacological approach is useful in guiding the detection of antifungal plants in Latin America mainly for infections in which the pathological expression is obvious and, therefore, the cure can be clearly observed.

Antifungal Activity of Eugenol Analogues. Influence of Different Substituents and Studies on Mechanism of Action

Twenty one phenylpropanoids (including eugenol and safrole) and synthetic analogues, thirteen of them new compounds, were evaluated for antifungal properties, first with non-targeted assays against a panel of human opportunistic pathogenic fungi. Some structure-activity relationships could be observed, mainly related to the influence of an allyl substituent at C-4, an OH group at C-1 and an OCH3 at C-2 or the presence of one or two NO2 groups in different positions of the benzene ring. All active compounds were tested in a second panel of clinical isolates of C. albicans and non-albicans Candida spp., Cryptococcus neoformans and dermatophytes. The eugenol derivative 4-allyl-2-methoxy-5-nitrophenol (2) was the most active structure against all strains tested, and therefore it was submitted to targeted assays. These studies showed that the antifungal activity of 2 was not reversed in the presence of an osmotic support such as sorbitol, suggesting that it does not act by inhibiting the fungal cell wall synthesis or assembly. On the other hand, the Ergosterol Assay showed that 2 did not bind to the main sterol of the fungal membrane up to 250 µg mL−1. In contrast, a 22% of fungal membrane damage was observed at concentrations = 1 × MIC and 71% at 4× MIC, when 2 was tested in the Cellular Leakage assay. The comparison of log P and MICs for all compounds revealed that the antifungal activity of the eugenol analogues would not to be related to lipophilicity.

Synthesis and antifungal activity of diverse C-2 pyridinyl and pyridinylvinyl substituted quinolines

Diverse 2-pyridinyl quinolines 6-12 and 2-pyridinilvinyl quinolines 13-17 were prepared using a straightforward synthesis based on the BiCl(3)-catalyzed multicomponent imino Diels-Alder (imino DA) reaction or a novel tandem imino DA/catalytic tetrahydroquinoline ring oxidation/Perkin condensation sequential process. All members of the series showed activities against dermatophytes and some of them possessed a broad spectrum of action. 2-(Pyridin-4-yl)quinoline 9 and 2-(2-pyridin-4-yl)vinyl)quinoline 16 showed the best MIC(80) and MIC(50) against the clinically important fungi Candida albicans and non-albicans Candida species. In turn, 6-ethyl-2-(pyridin-2-yl)quinoline 6 showed the best properties against standardized as well as clinical strains of Cryptococcus neoformans.

Synthesis of novel thiazole-based 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines as potential antitumor and antifungal agents.

A new series of novel thiazole-based 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines 6a-g and 7a-g were obtained with high regioselectivity from the reaction of triamino- or tetraaminopyrimidines 4 and 5 with α,β-unsaturated carbonyl compounds 3a-g based on 2,4-dichlorothiazol-5-carbaldehyde 1. Twelve of the synthesized compounds were selected and tested by US National Cancer Institute (NCI) for their antitumor activity against 60 different human tumor cell lines. Compounds 7d and 7g showed important GI50 ranges of 1.28-2.98 μM and 0.35-2.78 μM respectively under in vitro assays. In addition, 6a-g and 7a-g were tested for antifungal properties against the clinical important fungi Candida albicans and Cryptococcus neoformans. Although these compounds showed moderate activities against C. albicans, the 2-amino derivatives 7a-g and mainly 7a and 7b, showed high activity against standardized and clinical isolates of C. neoformans with MIC50 = 7.8-31.2 μg/mL, MIC80 = 15.6-31.2 μg/mL and MIC100 = 15.6-62.5 μg/mL. In addition, since both compounds were fungicide rather than fungistatic these thiazole-based 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines appear as good candidates for further development not only as antifungal but also as antitumor drugs.

Fungicide Activity of 5-(4-Chlorobenzylidene)-(Z)-2-dimethylamino-1,3-thiazol-4-one against Cryptococcus Neoformans

The present work describes the synthesis and antifungal evaluation of new 5‐arylidene‐(Z)‐2‐dimethylamino‐1,3‐thiazol‐4‐ones 4a–f, obtained by the reaction of aromatic aldehydes 1 and rhodanine 2 followed by treatment with DMF. All compounds were tested against a panel of yeasts, hialohyphomycetes, and dermatophytes using the microbroth dilution method. Compounds 4a and 4c showed antifungal activity, with compound 4a being the most active one. Compound 4a demonstrated to be fungicidal rather than fungistatic and selective activity against Cryptococcus neoformans and dermatophytes. MIC100, MIC80, and MIC50 of 4a were determined against a panel of clinical isolates of C. neoformans showing ranges of MICs between 2 and 16 μg/mL.

Synergistic mutual potentiation of antifungal activity of Zuccagnia punctata Cav. and Larrea nitida Cav. extracts in clinical isolates of Candida albicans and Candida glabrata.

BACKGROUND Zuccagnia punctata Cav. (Fabaceae) and Larrea nitida Cav. (Zygophyllaceae) are indistinctly or jointly used in traditional medicine for the treatment of fungal-related infections. Although their dichloromethane (DCM) extract have demonstrated moderate antifungal activities when tested on their own, antifungal properties of combinations of both plants have not been assessed previously. PURPOSE The aim of this study was to establish with statistical rigor whether Z. punctata (ZpE) and L. nitida DCM extract (LnE) interact synergistically against the clinically important fungi Candida albicans and Candida glabrata and to characterize the most synergistic combinations. STUDY DESIGN For synergism assessment, the statistical-based Boiks design was applied. Eight ZpE-LnE fixed-ratio mixtures were prepared from four different months of 1 year and tested against Candida strains. Lϕ (Loewe index) of each mixture at different fractions affected (ϕ) allowed for the finding of the most synergistic combinations, which were characterized by HPLC fingerprint and by the quantitation of the selected marker compounds. METHODS Lϕ and confidence intervals were determined in vitro with the MixLow method, once the estimated parameters from the dose-response curves of independent extracts and mixtures, were obtained. Markers (four flavonoids for ZpE and three lignans for LnE) were quantified in each extract and their combinations, with a valid HPLC-UV method. The 3D-HPLC profiles of the most synergistic mixtures were obtained by HPLC-DAD. RESULTS Three over four IC50ZpE/IC50LnE fixed-ratio mixtures displayed synergistic interactions at effect levels ϕ > 0.5 against C. albicans. The dosis of the most synergistic (Lϕ = 0.62) mixture was 65.96 µg/ml (ZpE = 28%; LnE = 72%) containing 8 and 36% of flavonoids and lignans respectively. On the other hand, one over four IC50ZpE/IC50LnE mixtures displays synergistic interactions at ϕ > 0.5 against C. glabrata. The dosis of the most synergistic (Lϕ = 0.67) mixture was 168.23 µg/ml (ZpE = 27%; LnE = 73%) with 9.7 and 31.6% of flavonoids and lignans respectively. CONCLUSIONS Studies with the statistical-based MixLow method, allowed for the finding of the most ZpE-LnE synergistic mixtures, giving support to a proper joint use of both antifungal herbs in traditional medicine.

Antifungal drugs combinations: a patent review 2000-2015.

ABSTRACT Introduction: Combination therapy has emerged as an approach to improve the efficacy of antifungal drugs. Its main objective is to achieve synergistic interaction with higher antifungal properties and lower toxic effects than each substance alone. Areas covered: Twenty-four patents disclosed in the period of 2000-2015 were covered in this review. Twenty of them were devoted to pharmacodynamic potentiation, while four were dedicated to pharmacokinetic actions. Expert opinion: The common characteristic of most patents published in this area is that the main partner is a commercial antifungal drug. In the most innovative combinations the second component was either a modifier of proton homeostasis, an antibody, an inhibitor of the adhesion of epithelial or endothelial cells or a keratinolytic agent that improves the skin penetration. The evaluation of synergism is always made with simple in vitro methods, which constitutes a weakness of the disclosed patents, due to the lack of in vivo studies, since the in vitro tests cannot predict the in vivo behavior. Also, it is surprising that none of the patents analyze the toxicity of the new combinations, taking into account that one of the main objectives of the combinations is to reduce the toxicity of the existing antifungal drugs.

Antifungal terpenoids from Hyalis argentea var. latisquama.

A detailed chemical study of the aerial parts and rhizomes of Hyalis argentea var. latisquama yielded a variety of sesqui- and diterpenes. In total, 26 compounds were isolated and identified, of which four are new, namely, two ent-kaurenes (1 and 2), a diterpene lactone (3), and a lindenanolide (4). The previously reported compounds included a series of lindenanolides, guaianolides, elemanolides, and additional diterpenes. The antifungal activity of the isolated compounds was tested against Cryptococcus neoformans and Candida albicans. Among the isolated compounds, the lindenanolides were the only structural class that showed strong antifungal activity, and onoseriolide acetate (5) was the most active. On the other hand, the isolated guaianolides were only moderately active, while the diterpenes did not show significant antifungal activity.

Hybrid combinations containing natural products and antimicrobial drugs that interfere with bacterial and fungal biofilms

BACKGROUND Biofilms contribute to the pathogenesis of many chronic and difficult-to eradicate infections whose treatment is complicated due to the intrinsic resistance to conventional antibiotics. As a consequence, there is an urgent need for strategies that can be used for the prevention and treatment of biofilm-associated infections. The combination therapy comprising an antimicrobial drug with a low molecular weight (MW) natural product and an antimicrobial drug (antifungal or antibacterial) appeared as a good alternative to eradicate biofilms. PURPOSE The aims of this review were to perform a literature search on the different natural products that have showed the ability of potentiating the antibiofilm capacity of antimicrobial drugs, to analyze which are the antimicrobial drugs most used in combination, and to have a look on the microbial species most used to prepare biofilms. RESULTS Seventeen papers, nine on combinations against antifungal biofilms and eight against antibacterial biofilms were collected. Within the text, the following topics have been developed: breaf history of the discovery of biofilms; stages in the development of a biofilm; the most used methodologies to assess antibiofilm-activity; the natural products with capacity of eradicating biofilms when acting alone; the combinations of low MW natural products with antibiotics or antifungal drugs as a strategy for eradicating microbial biofilms and a list of the low MW natural products that potentiate the inhibition capacity of antifungal and antibacterial drugs against biofilms. CONCLUSIONS AND PERSPECTIVES Regarding combinations against antifungal biofilms, eight over the nine collected works were carried out with in vitro studies while only one was performed with in vivo assays by using Caenorhabditis elegans nematode. All studies use biofilms of the Candida genus. A 67% of the potentiators were monoterpenes and sesquiterpenes and six over the nine works used FCZ as the antifungal drug. The activity of AmpB and Caspo was enhanced in one and two works respectively. Regarding combinations against bacterial biofilms, in vitro studies were performed in all works by using several different methods of higher variety than the used against fungal biofilms. Biofilms of both the gram (+) and gram (-) bacteria were prepared, although biofilm of Staphylococcus spp. were the most used in the collected works. Among the discovered potentiators of antibacterial drugs, 75% were terpenes, including mono, di- and triterpenes, and, among the atibacterial drugs, several structurally diverse types were used in the combinations: aminoglycosides, β-lactams, glucopeptides and fluoroquinolones. The potentiating capacity of natural products, mainly terpenes, on the antibiofilm effect of antimicrobial drugs opens a wide range of possibilities for the combination antimicrobial therapy. More in vivo studies on combinations of natural products with antimicrobial drugs acting against biofilms are highly required to cope the difficult to treat biofilm-associated infections.

Synthesis and antifungal activity of bile acid-derived oxazoles

Peracetylated bile acids (1a-g) were used as starting materials for the preparation of fourteen new derivatives bearing an oxazole moiety in their side chain (6a-g, 8a-g). The key step for the synthetic path was a Dakin-West reaction followed by a Robinson-Gabriel cyclodehydration. A simpler model oxazole (12) was also synthesized. The antifungal activity of the new compounds (6a-g) as well as their starting bile acids (1a-g) was tested against Candida albicans. Compounds 6e and 6g showed the highest percentages of inhibition (63.84% and 61.40% at 250 μg/mL respectively). Deacetylation of compounds 6a-g, led to compounds 8a-g which showed lower activities than the acetylated derivatives.