Mechanistic studies of Candida albicans photodynamic inactivation with Porophyllum obscurum hexanic extract and its isolated thiophenic compounds.

Porophyllum obscurum (Spreng) DC (Asteraceae) hexanic extract (PoHex) from aerial parts has demonstrated antifungal activity under UVA irradiation against Candida spp. isolates from patients with oropharyngeal candidiasis and four thiophenes were isolated as responsible of the activity. In the present work, we studied the photomechanisms whereby PoHex and their thiophenes produce photoinactivation of C. albicans. Reactive Oxygen Species generation by PoHex and thiophenes was evaluated: the production of superoxide anion, employing the NBT reduction assay; hydrogen peroxide, through the formation of a red quinoneimine; and singlet oxygen by using the 1,3-DPBF bleaching method. The action of ROS in fungal cells was investigated by evaluating binding of photosensitizer, leakage, apoptosis and stress sensibility that were performed by following M27-A3 guidelines, in parallel under "light" and "darkness" conditions. Results showed that the photosensitive antifungal activity of PoHex required oxygen and both type I (production of superoxide anion and hydrogen peroxide) and type II (production of singlet oxygen) reactions were involved. In addition, we found that ROS generated by PoHex did not cause release of cytoplasmic components due to membrane damage nor apoptosis of C. albicans. Treatment with PoHex and UVA increased cells sensitivities to osmotic stressors; did not reduce resistance to additional oxidative stress and possibly affected the structure of the cell wall. In addition, 2,2':5'2″terthiophene, the most active PS present in PoHex and the only one that generate single oxygen, at Minimal Fungicide Concentration, did not cause leakage nor apoptosis and did not increase sensitivities to osmotic and oxidative stressors. Results demonstrated that Photodynamic Inactivation employing PoHex under UVA does represent an alternative for topical antifungal therapy for oropharyngeal candidiasis.

Long-Lasting Primed State in Maize Plants: Salicylic Acid and Steroid Signaling Pathways as Key Players in the Early Activation of Immune Responses in Silks.

In the present study, we investigated the induced systemic resistance (ISR) activated by the beneficial fungus Trichoderma atroviride in maize plants, and the early immunological responses triggered after challenge with the ear rot pathogen Fusarium verticillioides. By transcriptional analysis, we were able to identify the gene core set specifically modulated in silks of maize plants expressing ISR. Our results showed that the main transcriptional reprogramming falls into genes involved in five main functional categories: cell structure or cell wall, amino acid and protein metabolism, stress responses, signaling, and transport. Among these ISR-related genes, it is important to highlight novel findings regarding hormone metabolism and signaling. The expression of hormone-dependent genes was in good agreement with the abscisic acid, jasmonic acid, and salicylic acid (SA) levels detected in the plants under study. The experimental design allowed the identification of novel regulatory elements related to a heightened state of defense in silks and suggests that steroids and SA are central components of a master regulatory network controlling the immunity of silks during ISR. The results presented also provide evidence about the molecular mechanisms used by maize silks against F. verticillioides to counteract pathogenic development and host invasion, including pathogenesis-related genes, plant cell-wall reinforcement, fungal cell-wall-degrading enzymes and secondary metabolism.

Plant phenolics and terpenoids as adjuvants of antibacterial and antifungal drugs.

BACKGROUND: The intensive use of antibacterial and antifungal drugs has dramatically increased the microbial resistance and has led to a higher number of difficult-to-eradicate infections. Combination therapy with two or more antimicrobial drugs has emerged some years ago to overcome the issue, but it has proven to be not completely effective. Natural secondary metabolites of MW ≤ 500 represent promising adjuvants for antimicrobials and have been the object of several researches that have increased in the last two decades. PURPOSE: The purpose of this Review is to do a literature search of the natural compounds that showed high enhancing capacity of antibacterials' and antifungals' effects against planktonic bacteria and fungi and to analyze which are the natural products most used in combination with a focus on polyphenols and terpenoids. RESULTS: One hundred of papers were collected for reviewing. Fifty six (56) of them deal with combinations of low MW natural products with antibacterial drugs against planktonic bacteria and forty four (44) on natural products with antifungal drugs against planktonic fungi. Of the antibacterial adjuvants, 41 (73%) were either polyphenols (27; 48%) or terpenes (14; 25%). The remaining 15 papers (27%), deal with different class of natural products. Since most natural potentiators belong to the terpene or phenolic structural types, a more detailed description of the works dealing with these type of compounds is provided here. Bacterial and fungal resistance mechanisms, the modes of action of the main classes of antibacterial and antifungal drugs and the methodologies most used to assess the type of interactions in the combinations were included in the Review too. CONCLUSIONS AND PERSPECTIVES: Several promising results on the potentiation effects of antifungals' and antibacterials' activities by low MW natural products mainly on polyphenols and terpenes were reported in the literature and, in spite of that most works included only in vitro assays, this knowledge opens a wide range of possibilities for the combination antimicrobial therapy. Further research including in vivo assays and clinical trials are required to determine the relevance of these antimicrobial enhancers in the clinical area and should be the focus of future studies in order to develop new antimicrobial combination agents that overpass the drawbacks of the existing antibiotics and antifungals in clinical use.

Antifungal photosensitive activity of Porophyllum obscurum (Spreng.) DC.: Correlation of the chemical composition of the hexane extract with the bioactivity.

We report Porophyllum obscurum as a source of new photosensitizers with potential use in Photodynamic Therapy as an alternative for oropharyngeal candidiasis treatment. The antifungal photosensitive activity of different extracts from P. obscurum was evaluated by using microdilution and bioautographic assays. The Minimum Fungicidal Concentration for hexanic extract under UV-A irradiation was 0.98µg/mL, but it was inactive in experiments without irradiation. The bioassay-guided fractionation of this extract led to the isolation of four thiophenes responsible for the photosensitive activity: 2,2':5'2″terthiophene, 5-(3-buten-1-ynyl)-2,2'-bithiophene, 5-(4-acetoxy-1-butenyl)-2,2'- bithiophene and 5-(4-hydroxy-1-butenyl)-2,2'- bithiophene, with Minimum Fungicidal Concentrations ranging 0.24-7.81µg/mL under UV-A irradiation. The activity of the hexanic extract was evaluated against 25 clinical strains of Candida spp. isolates as etiological agents of oropharyngeal candidiasis. No differences in susceptibility were observed in strains resistant and susceptible to conventional antifungal drugs. Qualitative and quantitative chemical analyses of seven samples of P. obscurum collected in four different phenological stages were carried out showing that full flowering stage possesses the highest thiophenes content. These data also allowed us to establish a correlation between the thiophene composition of the different extracts and their antifungal photosensitive activity, according to a second order polynomial model with the equation: y=11.2603-0.6831*x+0.0108*x2. The thiophenes isolated were the responsible of antifungal photosensitive activity and can be used for the future standardization of the extract. Results showed that P. obscurum hexanic extract could be potentially developed as an Herbal Medicinal Product to be applied as a photosensitizer in Photodynamic Therapy.

Antifungal drugs combinations: a patent review 2000-2015.

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.

Photodynamic inactivation of oropharyngeal Candida strains.

Oropharyngeal candidiasis (OPC) is an infection frequent in immunocompromised patients. Photodynamic therapy is an alternative to conventional treatments, based on the utilization of compounds that inhibit or kill microorganisms only under the effect of light, process known as Photodynamic Inactivation (PDI). In the present study, PDI of Candida spp. by the natural product α-terthienyl (α-T) was investigated following the guidelines of CLSI M27-A3, under UV-A light irradiation. The optimal values of two variables, exposure irradiation time (ET) and distance to the irradiation source (DIS) were established by employing Design Expert Software (DES). For this purpose, a panel of Candida strains isolated from OPC (C. albicans, C. tropicalis, C. parapsilosis and C. krusei) was employed and optimal values were 5 min (ET) and between 6.06 and 6.43 cm (DIS) with a desirability factor of 0.989. α-T plus UV-A light in the optimal conditions caused a complete reduction in viable cells in 5 min which was demonstrated by viable cells reduction assays and confocal microscopy after vital staining (propidium iodide/fluorescein diacetate). The germ tube formation of C. albicans was inhibited by α-T at sub-inhibitory concentrations. Results showed that α-T plus UV-A light could constitute an alternative for OPC treatments at the optimal conditions determined here.

Antibacterial, antifungal, phytotoxic, and genotoxic properties of two complexes of Ag(I) with sulfachloropyridazine (SCP): X-ray diffraction of [Ag(SCP)]n.

We report the synthesis, characterization, antibacterial and antifungal activities, phytotoxicity, and genotoxicity of two new complexes of silver(I) with sulfachloropyridazine (SCP), one of which is heteroleptic with SCP and SCN(-) ligands (Ag-SCP-SCN), the other of which is homoleptic (Ag-SCP); furthermore, the crystal structure of the homoleptic complex is disclosed. The heterocyclic N atom nearest to the Cl atom and the N(sulfonamide) atom could be coordination sites for the silver ion in the Ag-SCP-SCN complex. The Ag-SCP complex is a polymeric compound with metal-metal bonds, and the heterocyclic and sulfonamide N atoms are points of coordination for Ag(I) . Both complexes showed activity against all the tested bacteria, and in the cases of Escherichia coli and Pseudomonas aeruginosa, the action was better than that of SCP. In all cases, both silver-SCP complexes showed better antifungal activity than SCP, which was inactive against the tested fungi. Notably, the activity against P. aeruginosa, a nosocomial multidrug-resistant pathogen, was better than that of the reference antibiotic cefotaxim. Both silver-sulfa complexes displayed moderate activity against the tested yeast, especially for C. neoformans, which is an important fact considering the incidence of cryptococcosis, mainly in immune-deficient patients. No chromosomal aberrations were observed with the Allium cepa test, which is auspicious for further study of these complexes as potential drugs.

Effects of chirality on the antifungal potency of methylated succinimides obtained by Aspergillus fumigatus biotransformations. comparison with racemic ones.

Eighteen (3R) and (3R,4R)-N-phenyl-, N-phenylalkyl and N-arylsuccinimides were prepared with high enantioselectivity by biotransformation of maleimides with A. fumigatus. This environmentally friendly, clean and economical procedure was performed by the whole-cell fungal bioconversion methodology. Their corresponding eighteen racemic succinimides were prepared instead by synthetic methods. Both, the racemic and the chiral succinimides were tested simultaneously by the microbroth dilution method of CLSI against a panel of human opportunistic pathogenic fungi of clinical importance. Chiral succinimides showed higher antifungal activity than the corresponding racemic ones and the differences in activity were established by statistical methods. The bottlenecks for developing chiral drugs are how to obtain them through a low-cost procedure and with high enantiomeric excess. Results presented here accomplish both these objectives, opening an avenue for the development of asymmetric succinimides as new antifungal drugs for pharmaceutical use.