Functional textiles impregnated with biogenic silver nanoparticles from Bionectria ochroleuca and its antimicrobial activity.

Biogenic silver nanoparticles (AgNPs) were obtained throughout the fungal biosynthesis using extracellular filtrate of the epiphytic fungus B. ochroleuca and were incorporated in cotton and polyester fabrics by common impregnation procedure that was repeated once, twice or four times. Both fabrics were analyzed by scanning electron microscopy (SEM), and the effectiveness of impregnation was determined using inductively coupled plasma optical emission spectrometry (ICP OES). The AgNPs loaded fabrics showed potent antimicrobial activity on Staphylococcus aureus and Escherichia coli as well as on clinically relevant Candida albicans, Candida glabrata, and Candida parapsilosis, indicating that the AgNPs impregnation of cotton and polyester fabrics was efficient. AgNPs effectively inhibited the biofilm formation by Pseudomonas aeruginosa and was not toxic to Galleria mellonella larvae indicating a promising probability of biotechnological application.

Bactericidal, quorum quenching and anti-biofilm nanofactories: a new niche for nanotechnologists.

Despite several conventional potent antibacterial therapies, bacterial infections pose a significant threat to human health because they are emerging as the leading cause of death worldwide. Due to the development of antibiotic resistance in bacteria, there is a pressing demand to discover novel approaches for developing more effective therapies to treat multidrug-resistant bacterial strains and biofilm-associated infections. Therefore, attention has been especially devoted to a new and emerging branch of science "nanotechnology" to design non-conventional antimicrobial chemotherapies. A range of nanomaterials and nano-sized carriers for conventional antimicrobial agents have fully justified their potential to combat bacterial diseases by reducing cell viability, by attenuating quorum sensing, and by inhibiting/or eradicating biofilms. This communication summarizes emerging nano-antimicrobial therapies in treating bacterial infections, particularly using antibacterial, quorum quenching, and anti-biofilm nanomaterials as new approaches to tackle the current challenges in combating infectious diseases.

Investigation of local anesthetic and antimycobacterial activity of Ottonia martiana Miq. (Piperaceae).

Ottonia martiana is a plant popularly known in Brazil by the use for toothache. Ethanolic extract (EE), hexane fraction (HF), dichloromethane fraction (DF) and piperovatine obtained from O. martiana were assayed in vitro and in vivo. The acute toxicity of EE was determined, and LD50 values of 164.5 and 65.0 mg/kg by the oral and intraperitoneal routes, respectively, indicated a high toxicity for EE in vivo, explaining its popular use by topical administration only. A local anesthetic-like effect of EE and its fractions was observed in experimental models using pain induction, and such effect involved an analgesic action. The antimycobacterial activity of EE, HF, DF and piperovatine was evaluated against Mycobacterium tuberculosis H37Rv ATCC 27924. EE, HF, DF, and piperovatine showed a potential antimycobacterial effect with MICs of 16.0, 62.0, 62.0 and 8.0 µg/mL, respectively. Piperovatine was more effective than the EE or the other fractions. The selectivity index (SI=IC50/MIC) values calculated for EE, HF, DF and piperovatine based on the MICs and the cytotoxicity against J774 macrophages (IC50 by MTT assay) revealed values of 6.43, 2.34, 1.5 and 9.66, respectively.

Antifungal Activity of Mycogenic Silver Nanoparticles on Clinical Yeasts and Phytopathogens.

In this study, seven different silver nanoparticles (AgNPs) were obtained using the fungi species from the phylum Ascomycota, Aspergillus tubingensis, Aspergillus spp., Cladosporium pini-ponderosae, Fusarium proliferatum, Epicoccum nigrum, Exserohilum rostratum, and Bionectria ochroleuca, isolated from the Brazilian biodiversity, particularly from the mangrove and Caatinga biomes. The nanoparticles were coded as AgNP-AT, AgNP-Asp, AgNP-CPP, AgNP-FP, AgNP-EN, AgNP-ER, and AgNP-BO and characterized using spectrophotometry (UV-Vis), dynamic light scattering (DLS), zeta potential, transmission electron microcopy (TEM), and Fourier-transform infrared (FTIR) spectroscopy. All the AgNPs presented homogeneous size in the range from 43.4 to 120.6 nm (DLS) and from 21.8 to 35.8 nm (TEM), pH from 4.5 to 7.5, negative charge, and presence of protein coating on their surface. The antifungal activity of the AgNPs was evaluated on clinical strains of Candida albicans, and on the non-albicans species, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Candida guilliermondii, common in hospital infections, and against the phytopathogens Fusarium oxysporum, Fusarium phaseoli, Fusarium sacchari, Fusarium subglutinans, Fusarium verticillioides, and Curvularia lunata, which are species responsible for serious damage to agriculture production. The AgNPs were effective against the yeasts with MICs ranging from 1.25 to 40 µM and on the phytopathogens with MICs from 4 to 250 µM, indicating the promising possibility of application of these AgNPs as antifungal agents. The results indicated that the physicochemical parameters of the AgNPs, including the functional groups present on their surface, interfered with their antifungal activity. Overall, the results indicate that there is no specificity of the AgNPs for the yeasts or for the phytopathogens, which can be an advantage, increasing the possibility of application in different areas.

Effect of Monocerin, a Fungal Secondary Metabolite, on Endothelial Cells.

This study reports the isolation and identification of the endophytic fungus Exserohilum rostratum through molecular and morphological analysis using optical and transmission electron microscopy (TEM), as well as the procurement of its secondary metabolite monocerin, an isocoumarin derivative. Considering the previously observed biological activities of monocerin, this study was performed on human umbilical vein endothelial cells (HUVECs) that are widely used as an in vitro model for several different purposes. Important parameters, such as cell viability, senescence-associated ß-galactosidase, cellular proliferation by using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), apoptosis analysis with annexin, cellular morphology through scanning electron microscopy (SEM), and laser confocal analysis were evaluated after exposing the cells to monocerin. After 24 h of exposure to monocerin at 1.25 mM, there was more than 80% of cell viability and a low percentage of cells in the early and late apoptosis and necrosis. Monocerin increased cell proliferation and did not induce cell senescence. Morphological analysis showed cellular integrity. The study demonstrates aspects of the mechanism of action of monocerin on endothelial cell proliferation, suggesting the possibility of its pharmaceutical application, such as in regenerative medicine.

Effects of the antimycobacterial compound 2-phenoxy-1-phenylethanone on rat hepatocytes and formation of metabolites.

CONTEXT: Neolignans are usually dimers formed by oxidative coupling of allyl and propenyl phenols, and the neolignan analogue, 2-phenoxy-1-phenylethanone (LS-2) is a promising antimycobacterial compound showing very weak cytotoxicity in mammalian cells and lack of acute toxicity in murine models. OBJECTIVES: To investigate the mechanism of action of LS-2 in rat hepatocytes by evaluating the activity levels of enzymes related to oxidation status and drug-metabolizing activity. MATERIALS AND METHODS: Hepatocytes were treated with LS-2 from 0.05 up to 1 mM, for 24 and 48 h, and reduced glutathione (GSH), lipid peroxidation and cytochrome P450 enzyme (CYP450) activity were assayed. A homologous series of phenoxazone ethers were used as substrates to measure the enzymatic profile. The biotransformation of LS-2 was studied in hepatocytes by gas chromatography-mass spectrometry (GC-MS) for detection and analysis of possible metabolites. RESULTS: Hepatocytes treated with LS-2 up to 1 mM for 24 or 48 h did not induce the formation of GSH and lipid peroxidation. O-Dealkylation activities of the isoenzymes CYP4501A1, CYP4501A2, CYP4502B1 and CYP4502B2 were also not detected in the hepatocytes treated with LS-2 for 24 or 48 h. DISCUSSION AND CONCLUSION: The results indicate that LS-2 or its two detected metabolites, 2-phenoxy-1-phenylethanol and 2,4-(2-hydroxy-2-phenylethoxy)phenol, are not cytotoxic to rat hepatocytes. These compounds maintain a balance between the production of pro-oxidant agents and their respective antioxidant systems. The data show that enzymes related to oxidation status and drug-metabolizing activities are not involved in the mechanism of action of LS-2.

Overview of Bioactive Fungal Secondary Metabolites: Cytotoxic and Antimicrobial Compounds.

Microorganisms are known as important sources of natural compounds that have been studied and applied for different purposes in distinct areas. Specifically, in the pharmaceutical area, fungi have been explored mainly as sources of antibiotics, antiviral, anti-inflammatory, enzyme inhibitors, hypercholesteremic, antineoplastic/antitumor, immunomodulators, and immunosuppressants agents. However, historically, the high demand for new antimicrobial and antitumor agents has not been sufficiently attended by the drug discovery process, highlighting the relevance of intensifying studies to reach sustainable employment of the huge world biodiversity, including the microorganisms. Therefore, this review describes the main approaches and tools applied in the search for bioactive secondary metabolites, as well as presents several examples of compounds produced by different fungi species with proven pharmacological effects and additional examples of fungal cytotoxic and antimicrobial molecules. The review does not cover all fungal secondary metabolites already described; however, it presents some reports that can be useful at any phase of the drug discovery process, mainly for pharmaceutical applications.

The Bioflavonoids Rutin and Rutin Succinate Neutralize the Toxins of B. jararaca Venom and Inhibit its Lethality.

The venom of the Brazilian pit viper Bothrops jararaca (BjV) is a complex mixture of molecules, and snake venom metalloproteinases (SVMP) and serine proteinases (SVSP) are the most abundant protein families found therein. Toxins present in BjV trigger most of the deleterious disturbances in hemostasis observed in snakebites, i.e., thrombocytopenia, hypofibrinogenemia and bleedings. The treatment of patients bitten by snakes still poses challenges and the bioflavonoid rutin has already been shown to improve hemostasis in an experimental model of snakebite envenomation. However, rutin is poorly soluble in water; in this study, it was succinylated to generate its water-soluble form, rutin succinate (RS), which was analyzed comparatively regarding the chemical structure and characteristic features of rutin. Biological activities of rutin and RS were compared on hemostatic parameters, and against toxic activities of crude BjV in vitro. In vivo, C57BL/6 mice were injected i.p. with either BjV alone or pre-incubated with rutin, RS or 1,10-phenanthroline (o-phe, an SVMP inhibitor), and the survival rates and hemostatic parameters were analyzed 48 h after envenomation. RS showed the characteristic activities described for rutin - i.e., antioxidant and inhibitor of protein disulfide isomerase - but also prolonged the clotting time of fibrinogen and plasma in vitro. Differently from rutin, RS inhibited typical proteolytic activities of SVMP, as well as the coagulant activity of BjV. Importantly, both rutin and RS completely abrogated the lethal activity of BjV, in the same degree as o-phe. BjV induced hemorrhages, falls in RBC counts, thrombocytopenia and hypofibrinogenemia in mice. Rutin and RS also improved the recovery of platelet counts and fibrinogen levels, and the development of hemorrhages was totally blocked in mice injected with BjV incubated with RS. In conclusion, RS has anticoagulant properties and is a novel SVMP inhibitor. Rutin and RS showed different mechanisms of action on hemostasis. Only RS inhibited directly BjV biological activities, even though both flavonoids neutralized B. jararaca toxicity in vivo. Our results showed clearly that rutin and RS show a great potential to be used as therapeutic compounds for snakebite envenomation.

Wide visible-range activatable fluorescence ZnSe:Eu3+/Mn2+@ZnS quantum dots: local atomic structure order and application as a nanoprobe for bioimaging.

The development of QDs-based fluorescent bionanoprobe for cellular imaging fundamentally relies upon the precise knowledge of particle-cell interaction, optical properties of QDs inside and outside of the cell, movement of a particle in and out of the cell, and the fate of particle. We reported engineering and physicochemical characterization of water-dispersible Eu3+/Mn2+ co-doped ZnSe@ZnS core/shell QDs and studied their potential as a bionanoprobe for biomedical applications, evaluating their biocompatibility, fluorescence behaviour by CytoViva dual mode fluorescence imaging, time-dependent uptake, endocytosis and exocytosis in RAW 264.7 macrophages. The oxidation state and local atomic structure of the Eu dopant studied by X-ray absorption fine structure (XAFS) analysis manifested that the Eu3+ ions occupied sites in both ZnSe and ZnS lattices for the core/shell QDs. A novel approach was developed to relieve the excitation constraint of wide bandgap ZnSe by co-incorporation of Eu3+/Mn2+ codopants, enabling the QDs to be excited at a wide UV-visible range. The QDs displayed tunable emission colors by a gradual increase in Eu3+ concentration at a fixed amount of Mn2+, systematically enhancing the Mn2+ emission intensity via energy transfer from the Eu3+ to Mn2+ ion. The ZnSe:Eu3+/Mn2+@ZnS QDs presented high cell viability above 85% and induced no cell activation. The detailed analyses of QDs-treated cells by dual mode fluorescence CytoViva microscopy confirmed the systematic color-tunable fluorescence and its intensity enhances as a function of incubation time. The QDs were internalized by the cells predominantly via macropinocytosis and other lipid raft-mediated endocytic pathways, retaining an efficient amount for 24 h. The unique color tunability and consistent high intensity emission make these QDs useful for developing a multiplex fluorescent bionanoprobe, activatable in wide-visible region.

Antimicrobial Activity of Snake ß-Defensins and Derived Peptides.

ß-defensins are antimicrobial peptides presenting in vertebrate animals. They participate in innate immunity, but little is known about them in reptiles, including snakes. Although several ß-defensin genes were described in Brazilian snakes, their function is still unknown. The peptide sequence from these genes was deduced, and synthetic peptides (with approximately 40 amino acids and derived peptides) were tested against pathogenic bacteria and fungi using microbroth dilution assays. The linear peptides, derived from ß-defensins, were designed applying the bioisosterism strategy. The linear ß-defensins were more active against Escherichia coli, Micrococcus luteus, Citrobacter freundii, and Staphylococcus aureus. The derived peptides (7-14 mer) showed antibacterial activity against those bacteria and on Klebsiella pneumoniae. Nonetheless, they did not present activity against Candida albicans, Cryptococcus neoformans, Trychophyton rubrum, and Aspergillus fumigatus showing that the cysteine substitution to serine is deleterious to antifungal properties. Tryptophan residue showed to be necessary to improve antibacterial activity. Even though the studied snake ß-defensins do not have high antimicrobial activity, they proved to be attractive as template molecules for the development of antibiotics.

Differential physiological responses of a biogenic silver nanoparticle and its production matrix silver nitrate in Sorghum bicolor.

Silver nanoparticles (AgNP) have been extensively applied in different industrial areas, mainly due to their antibiotic properties. One of the environmental concerns with AgNP is its incorrect disposal, which might lead to severe environmental pollution. The interplay between AgNP and plants is receiving increasing attention. However, little is known regarding the phytotoxic effects of biogenic AgNP on terrestrial plants. This study aimed to compare the effects of a biogenic AgNP and AgNO3 in Sorghum bicolor seedlings. Seeds were germinated in increasing concentrations of a biogenic AgNP and AgNO3 (0, 10, 100, 500, and 1000 µM) in a growth chamber with controlled conditions. The establishment and development of the seedlings were evaluated for 15 days. Physiological and morpho-anatomical indicators of stress, enzymatic, and non-enzymatic antioxidants and photosynthetic yields were assessed. The results showed that both AgNP and AgNO3 disturbed germination and the establishment of sorghum seedlings. AgNO3 released more free Ag+ spontaneously compared to AgNP, promoting increased Ag+ toxicity. Furthermore, plants exposed to AgNP triggered more efficient protective mechanisms compared with plants exposed to AgNO3. Also, the topology and connectivity of the correlation-based networks were more impacted by the exposure of AgNO3 than AgNP. In conclusion, it is plausible to say that the biogenic AgNP is less toxic to sorghum than its matrix AgNO3.

Biogenic Aspergillus tubingensis silver nanoparticles' in vitro effects on human umbilical vein endothelial cells, normal human fibroblasts, HEPG2, and Galleria mellonella.

Silver nanoparticles (AgNPs) are widely incorporated into different hygiene, personal care, and healthcare products. However, few studies have been undertaken to determine the effects of biogenic AgNPs on human health. The effect of biosynthesized AgNPs using the fungus Aspergillus tubingensis culture was evaluated on human umbilical vein endothelial cells (HUVECs), normal human fibroblasts (FN1), human hepatoma cells (HEPG2) and a Galleria mellonella model. HUVECs were more susceptible to biogenic AgNPs than normal fibroblasts FN1 and intense cytotoxicity was observed only for very high concentrations at and above 2.5 µM for both cells. Normal human fibroblasts FN1 exposed to AgNPs for 24 h showed viability of 98.83 ± 8.40% and 94.86 ± 5.50% for 1.25 and 2.5 µM, respectively. At 5 and 10 µM, related to the control, an increase in cell viability was observed being 112.66 ± 9.94% and 117.86 ± 8.86%, respectively. Similar results were obtained for treatment for 48 and 72 h. At 1.25, 2.5, 5 and 10 µM of AgNPs, at 24 h, HUVECs showed 51.34 ± 7.47%, 27.01 ± 5.77%, 26.00 ± 3.03% and 27.64 ± 5.85% of viability, respectively. No alteration in cell distribution among different cycle phases was observed after HUVEC and normal fibroblast FN1 exposure to AgNPs from 0.01 to 1 µM for 24, 48 and 72 h. Based on the clonogenic assay, nanoparticles successfully inhibited HEPG2 cell proliferation when exposed to concentrations up to 1 µM. In addition to that, AgNPs did not induce senescence and no morphological alteration was observed by scanning electron microscopy on the endothelial cells. In the larvae of the wax moth, Galleria mellonella, a model for toxicity, AgNPs showed no significant effects, which corroborates to the safety of their use in mammalian cells. These results demonstrate that the use of A. tubingensis AgNPs is a promising biotechnological approach and these AgNPs can be applied in several biomedical situations.

Structure activity relationship, acute toxicity and cytotoxicity of antimycobacterial neolignan analogues.

OBJECTIVES: The study's aims were to evaluate the antimycobacterial activity of 13 synthetic neolignan analogues and to perform structure activity relationship analysis (SAR). The cytotoxicity of the compound 2-phenoxy-1-phenylethanone (LS-2, 1) in mammalian cells, such as the acute toxicity in mice, was also evaluated. METHODS: The extra and intracellular antimycobacterial activity was evaluated on Mycobacterium tuberculosis H37Rv. Cytotoxicity studies were performed using V79 cells, J774 macrophages and rat hepatocytes. Additionally, the in-vivo acute toxicity was tested in mice. The SAR analysis was performed by Principal Component Analysis (PCA). KEY FINDINGS: Among the 13 analogues tested, LS-2 (1) was the most effective, showing promising antimycobacterial activity and very low cytotoxicity in V79 cells and in J774 macrophages, while no toxicity was observed in rat hepatocytes. The selectivity index (SI) of LS-2 (1) was 91 and the calculated LD50 was 1870 mg/kg, highlighting the very low toxicity in mice. SAR analysis showed that the highest electrophilicity and the lowest molar volume are physical-chemical characteristics important for the antimycobacterial activity of the LS-2 (1). CONCLUSIONS: LS-2 (1) showed promising antimycobacterial activity and very weak cytotoxicity in cell culture, as well as an absence of toxicity in primary culture of hepatocytes. In the acute toxicity study there was an indication of absence of toxicity on murine models, in vivo.

Antimycobacterial brominated metabolites from two species of marine sponges.

A screening of 500 crude extracts of marine invertebrates against the growth of Mycobacterium tuberculosis H37Rv yielded MeOH extracts of the sponges Aplysina cauliformis and Pachychalina sp. with significant activity. Further bioassay-guided fractionation of both crude extracts led to the isolation of four bromine-containing metabolites. The known (+)-fistularin-3 (1) and 11-deoxyfistularin-3 (2), and the new compound 2-(3-amino-2,4-dibromo-6-hydroxyphenyl)acetic acid (3) were isolated from the sponge A. cauliformis, while the new bromotyrosine-derived 3-(3,5-dibromo-4-methoxyphenyl)-2-methoxy- N-methylpropan-1-ammonium (4) was isolated from Pachychalina sp. Compound 4 exhibited weak antimycobacterial activity while compounds 1-3 displayed activity against Mycobacterium tuberculosis H37Rv, with MICs of 7.1, 7.3 and 49 microM, respectively. Compounds 1 and 2 also exhibited low cytotoxicity against J744 macrophages, indicating that both 1 and 2 are interesting leads for the development of new anti-tuberculosis agents.