Anti-Candida property of a lignan glycoside derived from Styrax japonica S. et Z. via membrane-active mechanisms

Styraxjaponoside C was investigated with respect to its antifungal activity and mechanisms of action. Devoid of hemolytic activity, Styraxjaponoside C demonstrated an antifungal effect against the human pathogenic yeast Candida albicans in an energy-independent manner. To characterize the mechanisms of the antifungal activity of Styraxjaponoside C, fluorescence analysis with membrane probe 1,6-diphenyl-1,3,5-hexatriene, and flow cytometric analysis on C. albicans were conducted. The results showed that Styraxjaponosdie C induced cytoplasmic membrane perturbation. The current study suggested that Styraxjaponoside C was active against C. albicans with membrane-active mechanisms.     

Antifungal activity and mode of action of silver nano-particles on Candida albicans

In this study, the antifungal effects of silver nano-particles (nano-Ag) and their mode of action were investigated. Nano-Ag showed antifungal effects on fungi tested with low hemolytic effects against human erythrocytes. To elucidate the antifungal mode of action of nano-Ag, flow cytometry analysis, a glucose-release test, transmission electron microscopy (TEM) and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH), as a plasma membrane probe, were performed with Candida albicans. The results suggest nano-Ag may exert an antifungal activity by disrupting the structure of the cell membrane and inhibiting the normal budding process due to the destruction of the membrane integrity. The present study indicates nano-Ag has considerable antifungal activity, deserving further investigation for clinical applications. K.-J. Kim and W. S. Sung contributed equally to this work and should be considered co-first authors.     

Synthesis of Pogostone-Inspired Dehydroacetic Acid Derivatives and Their Antifungal Activity against Sclerotinia sclerotiorum (Lib.) de Bary

In an effort to exploit the natural antifungal pogostone, its simplified scaffold dehydroacetic acid (DHA) was used as a lead compound to semi-synthesize 56 DHA derivatives ( I1 – 48 , II , III , and IV1 – 6 ). Among them, compound IV4 exhibited the most potent antifungal activity with 11.0 μM EC₅₀ against mycelial growth of Sclerotinia sclerotiorum (Lib.) de Bary whose sclerotia production was also completely suppressed at this concentration. Furthermore, IV4 could completely inhibit infection cushion formation of S. sclerotiorum on rape leaves and achieved a preventive efficacy of 90.2 % at 500 μM, which was on the same level as that of commercial boscalid at 30 μM (88.7 %). The results of physiological and ultrastructural studies indicated that IV4 might disrupt the cell membrane permeability or induce the imbalance of mitochondrial membrane potential homeostasis to exert the antifungal mode of action. Besides, the robust and predicative three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed and discussed herein.     

Synthesis,structural characterization,in vitro DNA binding,and antitumor activity properties of Ru(II) compounds containing 2(2,6-dimethoxypyridine-3-yl)-1H-imidazo(4,5-f)[1, 10]phenanthroline

Abstract

The octahedral Ru(II) complexes containing the 2(2,6-dimethoxypyridine-3-yl)-1H-imidazo(4,5-f)[1, 10]phenanthroline ligand of type [Ru(N-N)₂(L)]²⁺, where N-N?=?phen (1,10-phenanthroline) (1), bpy (2,2^'-bipyridine) (2), and dmb (4,4^'-dimethyl-2,2^'-bipyridine) (3); L(dmpip) = (2(2,6-dimethoxypyridine-3-yl)1Himidazo(4,5-f)[1, 10]phenanthroline), have been synthesized and characterized by UV–visible absorption, molar conductivity, elemental analysis, mass, IR, and NMR spectroscopic techniques. The physicochemical properties of the Ru(II) complexes were determined by UV–Vis absorption spectroscopy. The DNA binding studies have been explored by UV–visible absorption, fluorescence titrations, and viscosity measurements. The supercoiled pBR322 DNA cleavage efficiency of Ru(II) complexes 1–3 was investigated. The antimicrobial activity of Ru(II) complexes was done against Gram-positive and Gram-negative microorganisms. The in vitro anticancer activities of all the complexes were investigated by cell viability assay, apoptosis, cellular uptake, mitochondrial membrane potential detection, and semi-quantitative PCR on HeLa cells. The result indicates that the synthesized Ru(II) complexes probably interact with DNA through an intercalation mode of binding with complex 1 having slightly stronger DNA binding affinity and anticancer activity than 2 and 3.     

Membrane-permeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate

Amphidinols, which are polyene-polyhydroxy metabolites produced by the marine dinoflagellate Amphidinium klebsii, possess potent antifungal and hemolytic activities. The membrane permeabilizing actions of amphidinol 3, the most potent homologue, were compared with those of polyene antibiotics, amphotericin B (AmB) and filipin, in hemolytic tests, ²³Na nuclear magnetic resonance (NMR)-based membrane permeabilizing assays, and UV spectroscopy for liposome-bound forms. In Na⁺ flux experiments using large unilamellar vesicles (LUVs), ion efflux by amphidinol 3 was inhibited by cholesterol or ergosterol, which was opposed to previous results [J. Mar. Biotechnol., 5 (1997) 124]. When the effect of the agents on the size of vesicles was examined by light scattering experiments, amphidinol 3 did not significantly alter their size while filipin and synthetic detergent Triton X-100 did. The observations implied that the activity of amphidinol 3 was mainly due to formation of large pores/lesions in liposomes rather than detergent-like disruption of membrane. The pore/lesion size was estimated to be 2.0-2.9 nm in diameter on the basis of osmotic protection experiments using blood cells. The UV spectra in liposomes, which revealed the close interaction of polyene moieties in a lipid bilayer, further implied that the membrane activity of amphidinol 3 is caused by the molecular assemblage formed in biomembrane. These results disclose that amphidinol 3 is one of few non-ionic compounds that possess potent membrane permeabilizing activity with non-detergent mechanism.     

In Vitro Susceptibility of <Emphasis Type="Italic">C. albicans</Emphasis> and <Emphasis Type="Italic">C. neoformens</Emphasis> to Potential Metabolites from <Emphasis Type="Italic">Streptomycetes</Emphasis>

Forty two Streptomycetes isolates from soils of Kodachadri region in Western ghats were recovered by soil dilution technique. Cross streak method was followed for primary screening of antifungal activity. Positive isolates were subjected to secondary screening by cold extraction of fermentation broth in butanol solvent. Six isolates exhibited broad spectrum antifungal activity against all the tested yeast pathogens like Candida albicans, Candida lipolytica, Cryptococcus neoformens and Saccharomyces cerevisiae. One isolate showed excellent antifungal activity against all test organisms with maximum zone of inhibition 60 mm each incase of C. neoformens and C. albicans. Partial characterization of antifungal metabolite by TLC resulted in a purple spot with an R_f value 0.50. The UV absorption spectra at 218 nm indicated possible chemical nature of the active metabolite as polyene group and purity was assessed by analytical HPLC.     

Low-Molecular-Weight,Biologically Active Compounds from Marine <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> Species

We have examined the ability of marine Proteobacteria from the Pseudoalteromonas genus and Alteromonas macleodii to produce low-molecular-weight, biologically active compounds with antimicrobial and surface-active properties. A new marine bacterium, Pseudoalteromonas issachenkonii, exhibited a high level of biological activity and produced antifungal and hemolytic compounds. A detailed spectroscopic investigation based on UV, IR, high-resolution mass spectrometry, and 2D ¹H and ¹³C nuclear magnetic resonance revealed that the former was indole-2,3-dione (isatin). The chemical structure of red-brown pigment (C₉H₇N₃OS₃) responsible for hemolytic activity remained unclear. Four of the 15 strains studied (P. luteoviolacea, P. rubra, P. undina, and P. issachenkonii) produced cell-bound, two (P. elaykovii and P. carrageenovora) produced extracellular, and one strain (P. citrea) produced cell-bound and extracellular fatty acids and phospholipids with surface activity. Neither peptides nor glycolipids with surface activity were detected.     

Improved recovery and biological activities of an engineered polyene NPP analogue in <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

NPP A1 produced by Pseudonocardia autotrophica is a unique disaccharide-containing polyene macrolide. NPP A1 was reported to have higher water solubility and lower hemolytic toxicity than nystatin A1 while retaining its antifungal activity. An engineered NPP A1 analogue, NPP A2, was generated by inactivation of the nppL gene, encoding a P450 monooxygenase in P. autotrophica. The resulting compound exhibited the corresponding chemical structure of NPP A1 but lacked a C10 hydroxyl group. In this study, newly developed crystallization recovery methods for NPP A2 purification, followed by an evaluation of in vitro antifungal activity and hemolytic activity, were performed. The crystallization methods were designed to eliminate the undesired viscous impurities encountered during the NPP A2 purification process, resulting in improved purity from 5.3 to 83.5% w/w. NPP A2 isolated from the improved purification process also exhibited two times higher antifungal activity and 1.8 times higher hemolytic toxicity than those of NPP A1. These results suggest that the minor structural modification of disaccharide-containing polyene macrolides, such as removing a C10 hydroxyl group, might require an alternative recovery process, such as crystallization, to confirm its improved biological activity.     

Antimycotic activities of Cinnamon-derived compounds against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.     

Membrane-permeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate

Amphidinols, which are polyene-polyhydroxy metabolites produced by the marine dinoflagellate Amphidinium klebsii, possess potent antifungal and hemolytic activities. The membrane permeabilizing actions of amphidinol 3, the most potent homologue, were compared with those of polyene antibiotics, amphotericin B (AmB) and filipin, in hemolytic tests, 23Na nuclear magnetic resonance (NMR)-based membrane permeabilizing assays, and UV spectroscopy for liposome-bound forms. In Na+ flux experiments using large unilamellar vesicles (LUVs), ion efflux by amphidinol 3 was inhibited by cholesterol or ergosterol, which was opposed to previous results [J. Mar. Biotechnol., 5 (1997) 124]. When the effect of the agents on the size of vesicles was examined by light scattering experiments, amphidinol 3 did not significantly alter their size while filipin and synthetic detergent Triton X-100 did. The observations implied that the activity of amphidinol 3 was mainly due to formation of large pores/lesions in liposomes rather than detergent-like disruption of membrane. The pore/lesion size was estimated to be 2.0-2.9 nm in diameter on the basis of osmotic protection experiments using blood cells. The UV spectra in liposomes, which revealed the close interaction of polyene moieties in a lipid bilayer, further implied that the membrane activity of amphidinol 3 is caused by the molecular assemblage formed in biomembrane. These results disclose that amphidinol 3 is one of few non-ionic compounds that possess potent membrane permeabilizing activity with non-detergent mechanism.     

Development of a plasma high-performance liquid chromatographic assay for LY303366, a lipopeptide antifungal agent, and its application in a dog pharmacokinetic study

An HPLC assay for plasma analysis of LY303366 (I), a semi-synthetic lipopeptide antifungal related to echinocandin B (ECB), was developed to support the selection and subsequent preclinical development of I. The method involved extraction of I from plasma with the aid of solid-phase extraction (SPE) cartidges followed by reversed-phase HPLC with UV detection at 300 nm. The method is simple, selective and is applicable to dog, rat, mouse and rabbit plasma. Validation studies using dog plasma showed that the values obtained for parameters of linearity, precision and accuracy were within acceptable limits. Based on analysis of 0.3 ml of plasma, the lower limit of quantitation was 20 ng/ml. The method has been successfully applied to determine the pharmacokinetic parameters of I in the dog following intravenous (i.v.) and oral administration. Compared to first generation ECB antifungal agents, the results of the i.v. dog study indicated a 50% reduction in clearance of the drug from plasma (0.1 l/h/kg) and an 18-fold increase in the volume of distribution at steady state (1.8 l/kg). When administered orally, compound I had an absolute bioavailability of 9%; however, plasma levels remained above the MIC for C. albicans (0.005 μg/ml) through 48 h. Given the excellent potency of I and its broad spectrum of activity relative to first generation ECB antifungal agents, the assay results for I indicate the potential for its use as a broad spectrum i.v. and oral antifungal agent.     

Metal based isatin-derived sulfonamides: Their synthesis,characterization, coordination behavior and biological activity

Some isatin derived sulfonamides and their transition metal [Co(II), Cu(II), Ni(II), Zn(II)] complexes have been synthesized and characterized. The structure of synthesized compounds and their nature of bonding have been inferred on the basis of their physical (magnetic susceptibility and conductivity measurements), analytical (elemental analyses) and spectral (IR, ¹H NMR and ¹³C NMR) properties. An octahedral geometry has been suggested for Co(II), Ni(II) and Zn(II) and square-planar for Cu(II) complexes. In order to assess the antibacterial and antifungal behavior, the ligands and their metal(II) complexes were screened for their in vitro antibacterial activity against four Gram-negative species, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa and Salmonella typhi and two Gram-positive species, Staphylococcus aureus and Bacillus subtilis and, for in vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata. In vitro cytotoxic properties of all the compounds were also studied against Artemia salina by brine shrimp bioassay. The results of average antibacterial/antifungal activity showed that zinc(II) complexes were found to be the most active against one or more bacterial/fungal strains as compared to the other metal complexes.     

Photosensitized reactions mediated by the major chromophore arising from glucose decomposition, result in oxidation and cross-linking of lens proteins and activation of the proteasome

Glucose solutions incubated at low oxygen concentration gave rise to the appearance of an absorption band in the UVA-visible region after 10 days. Further characterization evidenced that this band was composed by a single chomophore with maximum absorption bands at 335 and 365 nm. HPLC/MS and UV spectroscopy assays indicated that this product is composed by five unities of furan. Importantly, the presence of a compound with identical spectral and chromatographic properties was observed in the water-soluble fraction of cataractous human eye lenses. The photo-biological effects of this glucose-derived chromophore (GDC) have been addressed using targets of biological relevance, such as water-soluble proteins from eye lens and the proteasome present in this protein mixture. Increased protein oxidation and protein crosslinking was observed when lens proteins were exposed to UVA-visible light in the presence of GDC under a 5% and 20% oxygen atmosphere. In addition, an increased proteasome peptidase activity was also observed. However, the use of D₂O resulted in decreased proteasome activity, suggesting that singlet oxygen promotes the impairment of proteasome activity. Our results suggest that the species generated by Type I and Type II mechanisms have opposite effects on proteasome activity, being Type I a positive activator while Type II lead to impairment of proteasome function.     

Evidence of excited state absorption in PS II membrane fragments

Utilizing a two-beam technique in the frequency domain, the pumped absorption of PS II membrane fragments from spinach and of acetonic chlorophyll-a solutions was measured at room temperature. In a very narrow wavelength region (0.2 nm around the pump pulse wavelength) the relative test beam transmission exhibited either a decrease or an increase, respectively, dependent on the intensity of a strong pump beam. In contrast, the transmission changes of chl-a solutions were not affected by the wavelength mistuning between pump and test beam. The data obtained for PS II membrane fragments were interpreted in terms of excited state absorption of pigment-protein clusters within the light-harvesting complex of PS II. The interpretation of the small absorption band as a homogeneously broadened line led to a transversal relaxation time for chlorophyll in vivo of about 1 ps.Abbreviations PS I photosystem I of green plants - PS II photosystem II of green plants - P700 primary donor of PS I - P680 primary donor of PS II     

Purification and Some Properties of Two Types of Penicillium Lipase,I and II,and Conversion of Types I and II under Various Modification Conditions

The lipase produced by a strain of Penicillium crustosum Thom was fractionated into three lipase components, I～III by DEAE cellulose column chromatography, and two of them, I and II were purified and obtained in crystalline form respectively, which proved homogeneous by electrophoresis and ultracentrifugal analysis. Lipase I was an ordinary lipase with molecular weight about 29,000 hydrolyzing olive oil and tributyrin favourably in almost the same degree, while II, rather, a so-called tributyrinase with M. W. about 32,000 hydrolyzing tributyrin more efficiently than olive oil. The site of the activity on olive oil in these lipase was generally sensitive to sodium desoxycholate, ethylenediamine-tetraacetate (EDTA), and p-chloromercuribenzoate (PCMB), and lipase I was converted to a lipase II by a treatment with these reagents. Also, partial degradation of I by proteinase (‘pronase’) yielded the enzyme fragment of type II. On the other hand, treatment of the enzymes with hydrogen peroxide or sodium borohydride caused the conversion of type II into I. From the observation of UV difference spectrum during incubation with sodium desoxycholate it was indicated that the situation of tryptophane residue in enzyme molecule may have a significance in the activity of lipase I on olive oil.     

In-vitro Antibacterial,Antifungal and cytotoxic activity of cobalt (II), copper (II), nickel (II) and zinc (II) complexes with furanylmethyl- and thienylmethyl-dithiolenes: [1, 3-dithiole- 2-one and 1,3-dithiole-2-thione]

Some antibacterial and antifungal furanylmethyl-and thienylmethyl dithiolenes and, their Co(II), Cu(II), Ni (II) and Zn (II) complexes have been synthesized, characterized and screened for their in vitro antibacterial activity against four Gram-negative; Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi and Shigella flexeneri, and two Gram-positive; Bacillus subtilis and Staphylococcus aureus bacterial strains, and for in-vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata. All compounds showed significant antibacterial and antifungal activity. The metal complexes, however, were shown to possess better activity as compared to the simple ligands. The brine shrimp bioassay was also carried out to study their in-vitro cytotoxic properties.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Antifungal effect and mode of action of glochidioboside against Candida albicans membranes

Glochidioboside was obtained from Sambucus williamsii and its biological effect has not been reported. Its antifungal activity against pathogenic fungi and the mode of action involved in its effect were examined. Glochidioboside exerted antifungal effect with almost no hemolytic effect against human erythrocytes. To understand its antifungal mechanisms, membrane studies were done. Using two dyes, 3,3′-dipropylthiacarbocyanine iodide [DiSC₃(5)] and propidium iodide, membrane depolarization and permeabilization by glochidioboside were confirmed. Furthermore, the membrane-active mechanism was proven by synthesizing a model membrane, calcein-encapsulating large unilamellar vesicles (LUVs), and also by observing the influx of different sized fluorescent dyes, such as calcein, FD4 and FD10, into the fungal cells. The membrane-active action was pore-forming action with radii between 1.4 and 2.3 nm. Finally, three dimensional (3D) flow cytometric analysis showed the shrinkage of the fungal cells from the membrane damage. In conclusion, this study suggests that glochidioboside exerts an antifungal activity through a membrane-disruptive mechanism.     

Novel fluconazole derivatives with promising antifungal activity

The fungistatic nature and toxicity concern associated with the azole drugs currently on the market have resulted in an increased demand for new azole antifungal agents for which these problematic characteristics do not exist. The extensive use of azoles has resulted in fungal strains capable of resisting the action of these drugs. Herein, we report the synthesis and antifungal activity of novel fluconazole (FLC) analogues with alkyl-, aryl-, cycloalkyl-, and dialkyl-amino substituents. We evaluated their antifungal activity by MIC determination and time-kill assay as well as their safety profile by hemolytic activity against murine erythrocytes as well as cytotoxicity against mammalian cells. The best compounds from our study exhibited broad-spectrum activity against most of the fungal strains tested, with excellent MIC values against a number of clinical isolates. The most promising compounds were found to be less hemolytic than the least hemolytic FDA-approved azole antifungal agent voriconazole (VOR). Finally, we demonstrated that the synthetic alkyl-amino FLC analogues displayed chain-dependent fungal membrane disruption as well as inhibition of ergosterol biosynthesis as possible mechanisms of action.     

Morphological changes of <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic">oxysporum</Emphasis> induced by CF66I,an antifungal compound from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis>

The morphological effects of CF66I, an antifungal compound produced by Burkholderia cepacia, on growing hyphae of Fusarium oxysporum were studied by fluorescence microscopy (FM) and transmission electron microscopy (TEM). At 20 μg/ml, CF66I strongly inhibited growth and induced significant changes of the hyphal morphology. These changes included swelling of hyphae with considerable thickening cell wall and abnormal chitin deposition, which was indicative of the alterations in cell wall structure. Furthermore, fluorescein diacetate (FDA) staining indicated the loss of intracellular esterase activity. CF66I probably inhibits fungal growth by interfering with the cell metabolic pathways. At 120 μg/ml, CF66I killed F. oxysporum (accompanied by propidium iodide permeation, intracellular cytoplasm leakage and crushing of hyphal tips), probably by direct damage to the cell membrane. Thus, there are two different antifungal mechanisms of CF66I, depending on its concentration, and further studies on this compound might be useful for us to develop a new class of antifungal agents.