Facile Synthesis of Bio-Antimicrobials with "Smart" Triiodides.

Multi-drug resistant pathogens are a rising danger for the future of mankind. Iodine (I2) is a centuries-old microbicide, but leads to skin discoloration, irritation, and uncontrolled iodine release. Plants rich in phytochemicals have a long history in basic health care. Aloe Vera Barbadensis Miller (AV) and Salvia officinalis L. (Sage) are effectively utilized against different ailments. Previously, we investigated the antimicrobial activities of smart triiodides and iodinated AV hybrids. In this work, we combined iodine with Sage extracts and pure AV gel with polyvinylpyrrolidone (PVP) as an encapsulating and stabilizing agent. Fourier transform infrared spectroscopy (FT-IR), Ultraviolet-visible spectroscopy (UV-Vis), Surface-Enhanced Raman Spectroscopy (SERS), microstructural analysis by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and X-Ray-Diffraction (XRD) analysis verified the composition of AV-PVP-Sage-I2. Antimicrobial properties were investigated by disc diffusion method against 10 reference microbial strains in comparison to gentamicin and nystatin. We impregnated surgical sutures with our biohybrid and tested their inhibitory effects. AV-PVP-Sage-I2 showed excellent to intermediate antimicrobial activity in discs and sutures. The iodine within the polymeric biomaterial AV-PVP-Sage-I2 and the synergistic action of the two plant extracts enhanced the microbial inhibition. Our compound has potential for use as an antifungal agent, disinfectant and coating material on sutures to prevent surgical site infections.

Antifungal Activity of Capric Acid, Nystatin, and Fluconazole and Their In Vitro Interactions Against Candida Isolates from Neonatal Oral Thrush.

Due to the increasing resistance of various Candida species to azole drugs, particularly fluconazole, it would be of significant importance to look for alternative therapies. The aim of this study was to investigate the antifungal activity of capric acid and its in vitro interactions with nystatin and fluconazole against Candida isolates. A total of 40 Candida isolates (C. albicans, 36; C. kefyr, 2; C. tropicalis, 1; C. glabrata, 1) collected from the oral cavity of neonates with oropharyngeal candidiasis and a reference strain of C. albicans (ATCC 10231) were used in this study. Antifungal activity of capric acid and two comparator antifungal drugs, namely fluconazole and nystatin, was tested according to CLSI M27-A3/M60 method. The in vitro interaction between capric acid with fluconazole and nystatin was determined following a checkerboard method and results were interpreted using fractional inhibitory concentration index. Nystatin had the lowest minimum inhibitory concentrations (range, 0.125-8 µg/mL; geometric mean [GM], 0.6229 µg/mL) followed by fluconazole (range, 0.5-16 µg/mL; GM, 1.9011 µg/mL) and capric acid (range, 128-2,048 µg/mL; GM, 835.9756 µg/mL). When tested in combination, capric acid with fluconazole demonstrated synergistic, indifferent, and antagonistic interactions in 3 (7.317%), 24 (58.536%), and 14 (34.146%) cases, respectively. For combination of capric acid with nystatin, synergistic, indifferent, and antagonistic interactions were observed in 1 (2.439%), 19 (46.341%), and 21 (51.219%) cases, respectively. All cases of synergistic interactions were against resistant or susceptible dose-dependent isolates. Fluconazole, nystatin, and capric acid seem to be more effective when they are used alone compared with their combination. However, their combination might be effective on resistant isolates.

Formulation, characterization and in vitro evaluation of antifungal activity of Nystatin micro emulsion for topical application.

The current research aims at development and assessment of o/w nystatin microemulsion. The pseudoternary phase diagrams were developed to determine microemulsion existence regions by water titration method. Nystatin was liquefied in the blend of oil phase, surfactant and cosurfactant. Microemulsion was made by deliberate mixing of water and stirring in this blend. The S-mix (surfactant-cosurfactant mixtures) of the ratio 1:2 was found better than 1:1 and 2:1 S-mix ratios. In vitro permeation studies by Franz diffusion cell revealed faster rate of nystatin release from such microemulsion (5.37µg/cm2/h) as compared to nystrin (4.79µg/cm2/h), a commercially available aqueous suspension. Kinetic modeling demonstrated zero order drug release and release mechanism found to be anomalous i.e. superposition of dispersion and swelling controlled drug release. Antifungal activity was performed using well diffusion method in vitro against Candida albicans cultures grown on Sabouraud's dextrose agar. The results also confirmed the high diffusion rate of drug from microemulsion as compared to aqueous suspension. The outcomes of this study propose that topical microemulsion of nystatin provides better antifungal activity as compared to emulsion gels or aqueous suspensions.

Identification of multi-targeted anti-migraine potential of nystatin and development of its brain targeted chitosan nanoformulation.

The complex pathophysiology involved in migraine necessitates the drug treatment to act on several receptors simultaneously. The present investigation was an attempt to discover the unidentified anti-migraine activity of the already marketed drugs. Shared featured pharmacophore modeling was employed for this purpose on six target receptors (ß2 adrenoceptor, Dopamine D3, 5HT1B, TRPV1, iGluR5 kainate and CGRP), resulting in the generation of five shared featured pharmacophores, which were further subjected to virtual screening of the ligands obtained from Drugbank database. Molecular docking, performed on the obtained hit compounds from virtual screening, indicated nystatin to be the only active lead against the receptors iGluR5 kainate receptor (1VSO), CGRP (3N7R), ß2 adrenoceptor (3NYA) and Dopamine D3 (3PBL) with a high binding energy of -11.1, -10.9, -10.2 and -12kcal/mole respectively. The anti-migraine activity of nystatin was then adjudged by fabricating its brain targeted chitosan nanoparticles. Its brain targeting efficacy, analyzed qualitatively by confocal laser scanning microscopy, demonstrated a significant amount of drug reaching the brain. The pharmacodynamic models on Swiss male albino mice revealed significant anti-migraine activity of the nanoformulation. The present study reports for the first time the therapeutic potential of nystatin in migraine management, hence opening avenues for its future exploration.

Cryptic antifungal compounds active by synergism with polyene antibiotics.

The majority of antifungal compounds reported so far target the cell wall or cell membrane of fungi, suggesting that other types of antibiotics cannot exert their activity because they cannot penetrate into the cells. Therefore, if the permeability of the cell membrane could be enhanced, many antibiotics might be found to have antifungal activity. We here used the polyene antibiotic nystatin, which binds to ergosterol and forms pores at the cell membrane, to enhance the cellular permeability. In the presence of nystatin, many culture extracts from entomopathogenic fungi displayed antifungal activity. Among all the active extracts, two active components were purified and identified as helvolic acid and terramide A. Because the minimum inhibitory concentration of either compound was reduced four-fold in the presence of nystatin, it can be concluded that this screening method is useful for detecting novel antifungal activity.

Innovative formulation of nystatin particulate systems in toothpaste for candidiasis treatment.

Oral candidiasis is a mycosis on the mucous membranes of the mouth but not limited to the mouth. Nystatin is one of the most frequently employed antifungal agents to treat infections and may be safely given orally as well as applied topically but its absorption through mucocutaneous membranes such as the gut and the skin is minimal. The purpose of this study is to enhance the effectiveness of nystatin using particulate system such as beads, micro- and nanoparticles of alginate incorporated into toothpaste. Those particulate systems of nystatin were prepared by extrusion/external gelation for beads and emulsification/internal gelation for micro- and nanoparticles and characterized. Small, anionic charged and monodispersed particles were successfully produced. The type of particulate system influenced all previous parameters, being microparticles the most suitable particulate system of nystatin showing the slowest release, the highest inhibitory effect of Candida albicans over a period of one year. Those results allowed the conclusion that alginate exhibits properties that enable the in vitro functionality of encapsulated nystatin and thus may provide the basis for new successful approaches for the treatment of oral antifungal infections such as oral candidiasis.

The interaction of polysaccharide from Auricularia polytricha with quantum dots and the protection of plasmid DNA from damage.

As a kind of folk medicine, edible mushrooms are known to be of medicinal characteristics, for example antitumor activity. However, the mechanism is not clear. In this study, the fluorescence (FL) spectroscopy was used to study the binding reactions of polysaccharide extracted from Auricularia polytricha with quantum dots (QDs). The modified Stern-Volmer quenching constant at different temperatures and corresponding thermodynamic parameters were calculated (∆G < 0, ∆H < 0, and ∆S < 0). The results show that the quenching constant is inversely correlated with temperature. It indicates that the quenching mechanism is the static quenching in nature rather than dynamic quenching. The negative values of free energy (∆G < 0) suggest that the binding process is spontaneous; ∆H < 0 and ∆S < 0 suggest that the binding of polysaccharide to QDs is enthalpy-driven. QDs were found to damage the plasmid DNA according to atomic force microscopy images. DNA damage is the important factor to induce tumor. Many chemical substances can induce tumor, which has been proved by modern medical science. In the presence of polysaccharide from A. polytricha, however, DNA was protected from damage due to polysaccharide winding around QDs, which is the basis for the bioeffect of polysaccharides. This study helps to understand that antitumor activity of edible mushrooms is attributed to protection of DNA from damage in the presence of harmful substances.

Mechanism of activity and toxicity of Nystatin-Intralipid.

A novel lipid formulation of Nystatin (NYT), Nystatin-Intralipid (NYT-IL), which was found to be more active and less toxic in vitro and in vivo, was developed in our laboratory. The aim of the present study was to explore the possible mechanisms underlying its biological activity. To assess mechanisms affecting fungal cells we conducted the following experiments: killing kinetics, scanning and transmission electron microscopy (EM), measurements of potassium ion leakage and susceptibility in the presence of ergosterol. To study mechanisms affecting mammalian cells, we evaluated the effect of NYT-IL on a kidney cell line, with respect to viability, metabolic activity, potassium leakage and internalization of FITC-labeled human transferrin. NYT-IL exhibited killing kinetics patterns against Candida albicans similar to those of NYT and caused disruption of fungal cells and potassium ion leakage. Susceptibility tests showed that NYT-IL had lower antifungal activity in the presence of ergosterol. Thus, NYT-IL acts apparently by damaging fungal membrane, possibly through interaction with ergosterol, and maybe by additional modes of action. NYT-IL did not cause potassium leakage from mammalian kidney cells at any tested concentration and was not cytotoxic, whereas NYT, at high concentrations, led to K(+) leakage and was cytotoxic. Furthermore, the high NYT concentration interfered in the internalization process of human transferrin receptor (hTfnR) while NYT-IL did not. In summary, the Intralipid formulation of NYT diminishes the mechanisms responsible for toxicity to mammalian cells but preserves mechanisms of action against fungi, thereby suggesting superiority of NYT-IL as compared to NYT as an antifungal agent.

Activity of an Intralipid formulation of nystatin in murine systemic candidiasis.

Since nystatin (NYT) is used only topically owing to its toxicity upon systemic administration, a study was initiated aiming to develop a formulation of NYT that could be used systemically against invasive mycoses. The present research is a continuation of previous in vitro investigation of the antifungal effect of nystatin-Intralipid (NYT-IL) against Candida, exploring its in vivo activity. NYT-IL was tested in murine systemic candidiasis induced in naïve as well as cyclophosphamide-immunosuppressed female ICR mice. The infection was assessed by survival rate (SR), mean survival time (MST) and qualitative and quantitative fungal organ colonisation. Mice were treated by intravenous administration of various doses of NYT-IL for 5 consecutive days starting either 24h or 48 h after the initiation of infection. The experiments showed that NYT-IL is therapeutically effective in the murine candidiasis model. NYT-IL was found to be less toxic in vivo than NYT and therefore higher doses of NYT-IL could be used. The efficacy of NYT-IL was expressed in treated naïve and immunosuppressed mice by increased SR, prolonged MST and reduced fungal organ colonisation. Early initiation of treatment increased efficacy. In summary, the Intralipid formulation of NYT can be administered parenterally and is effective against systemic experimental Candida infection.

Nystatin-intralipid preparation: characterization and in vitro activity against yeasts and molds.

The objective of our studies is the development of a novel formulation of nystatin (NYT) that could be administered systemically and might be used for therapy of invasive mycoses. We developed a formulation of nystatin and intralipid (IL), which is a clinically used food supplement, and this report focuses on the characterization of NYT-IL, assessment of its antifungal activity and in vitro toxicity. We characterized physical properties of the NYT-IL preparation and its stability during storage. Susceptibility of Candida, Aspergillus and Fusarium species was determined using a CLSI technique. In vitro toxicity of NYT-IL was assessed using an assay measuring hemolysis of sheep red blood cells (SRBC) and leakage of potassium. It was found that: (1) the particle size in NYT-IL did not differ from that of IL; (2) over 80% of NYT was in association with IL; and (3) these features did not change during storage. All Candida and Aspergillus strains had lower minimal inhibitory concentration (MIC) values for NYT-IL than that for NYT; the MICs of the Fusarium strains were similar for NYT & NYT-IL. Toxicity assays showed that the NYT-IL formulation is less toxic than NYT. In conclusion, we describe a novel, characterized, stable formulation of nystatin, nystatin-intralipid, with in vitro activity against pathogenic Candida and Aspergillus species.

Use of tuftsin bearing nystatin liposomes against an isolate of Candida albicans showing less in vivo susceptibility to amphotericin B.

In the present study, we evaluated tuftsin bearing nystatin liposomes for their potential against an isolate of Candida albicans (C. albicans) showing less in vivo susceptibility to amphotericin B (Amp B). The liposomised-Amp B in higher doses was found to be effective in elimination of less susceptible strain of C. albicans (C. albicans JMCR) in Balb/c mice, but may not be recommended due to toxicity constraints. On the other hand, liposomal nystatin was shown to possess higher efficacy as compared to that of Amp B, and was pertinent in treatment of C. albicans JMCR strain. The data of present work reveals that the incorporation of nystatin in tuftsin-bearing-liposomes results in a significant increase in its efficacy against experimental murine candidiasis. Interestingly, the pre-treatment of animals with liposomised-tuftsin prior to challenge with C. albicans infection was more effective in elimination of the pathogen from host and shows an advantage in prophylactic perspectives.

Double-layered mucoadhesive tablets containing nystatin.

The objective of this work was to design a mucoadhesive tablet with a potential use in the treatment of oral candidosis. A 2-layered tablet containing nystatin was formulated. Lactose CD (direct compression), carbomer (CB), and hydroxypropylmethylcellulose (HPMC) were used as excipients. Tablets were obtained through direct compression. Properties such as in vitro mucoadhesion, water uptake, front movements, and drug release were evaluated. The immediate release layer was made of lactose CD (100 mg) and nystatin (30 mg). The CB:HPMC 9:1 mixture showed the best mucoadhesion properties and was selected as excipient for the mucoadhesive polymeric layer (200 mg). The incorporation of nystatin (33.3 mg) in this layer affected the water uptake, which, in turn, modified the erosion front behavior. Nystatin showed a first-order release. The polymeric layer presented an anomalous kinetic (n = 0.82) when this layer was individually evaluated. The mucoadhesive tablet formulated in this work releases nystatin quickly from the lactose layer and then in a sustained way, during approximately 6 hours, from the polymeric layer. The mixture CB:HPMC 9:1 showed good in vitro mucoadhesion. A swelling-diffusion process modulates the release of nystatin from this layer. A non-Fickian (anomalous) kinetic was observed.

Role of the sterol superlattice in the partitioning of the antifungal drug nystatin into lipid membranes.

Nystatin isolated from Streptomyces is a polyene antibiotic that is frequently used in the treatment and prophylaxis of fungal infections. Here, the fractional sterol concentration dependencies of the partition coefficient for partitioning of nystatin into ergosterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), cholesterol/DMPC, ergosterol/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC), and ergosterol/POPC/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylethano lam ine (POPE) multilamellar vesicles have been determined fluorometrically at 37 degrees C using approximately 0.3-1.0 mol % sterol concentration increments over a wide concentration range (e.g., 18-54 mol % sterol). This unconventional approach of varying membrane sterol content, in contrast to previous studies using large sterol concentration increments (e.g., 10 mol %), leads to a striking observation. The partition coefficient of nystatin changes dramatically with membrane sterol content in a well-defined alternating manner, displaying a local minimum at or very close to the critical sterol mole fractions (e.g., 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % sterol) predicted for sterols regularly distributed in either hexagonal or centered rectangular superlattices. In ergosterol/DMPC bilayers, for example, there is a >3-fold increase in nystatin partitioning with a minute change (approximately 1 mol %) in sterol content on either side of the critical sterol mole fraction, 25.0 mol %. These results provide semifunctional evidence supporting the sterol regular distribution model [Chong, P. L.-G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10069-10073]. More importantly, these results reveal a new membrane phenomenon, that is, that nystatin partitioning is affected by the extent of sterol regular distribution in the plane of the membrane. This phenomenon occurs not only in saturated (e.g., DMPC) but also in unsaturated (e.g., POPC) lipid membranes, and persists in the presence of polar headgroup heterogeneity (e.g., POPC/POPE). This membrane property points to a new method for studying the interactions of polyene antibiotics with sterol-containing membranes, and the need to consider the membrane sterol content of the target cells when administering nystatin or other polyene antibiotics.

Association of polyene antibiotics with sterol-free lipid membranes. II. Hydrophobic binding of nystatin to dilauroylphosphatidylcholine bilayers.

Interaction of nystatin A1 with multilamellar vesicles (MLV) of dilauroylphosphatidylcholine (DLPC), observed either by adding nystatin to preformed MLV (mixtures I) or by incorporating it during the formation of vesicles (mixtures II, inner lamellas of MLV in contact with nystatin) was investigated for 0.002 < or = nystatin/DLPC = R(A) < or = 0.20, by four complementary methods. The main results were: (i) Ultraviolet absorption and circular dichroism (CD) spectra of mixtures I revealed the occurrence of a saturable association with a stoichiometry (R(A) = 0.007 +/- 0.002) constant between 3 and 33 degrees C. (ii) By differential scanning calorimetry, thermograms of the two types of mixtures were similar only when water was in great excess. In the opposite (e.g., (H2O)/(DLPC) = R(W) < or = 300), mixture II thermograms displayed two features, upshifted by about 6.5 degrees C with respect to the sharp peak observed with mixture I, resembling those obtained for pure DLPC when the low-temperature phase was the subgel phase. For this R(W), the nystatin absolute concentrations were those for which nystatin form superaggregates as revealed by the nystatin CD spectra. It is proposed that these superaggregates are excluded from the interlamellar spacings of MLV and exert a pumping action on the interlamellar water. The subsequent dehydration of the inner lamellas is thought to convert them into the subgel state. (iii) 2H-NMR spectra of sn-2-perdeuterated DLPC MLV + nystatin mixtures II, confirmed such a temperature shift of the main transition. They showed, in addition, an ordering of the aliphatic chains immediately above the transition temperature, equivalent to a bilayer thickening of 2 A.

An in vitro analysis of a sustained release system for the treatment of denture stomatitis.

This study investigated the feasibility of a sustained-release delivery system for the treatment of denture stomatitis using four antifungal agents (chlorhexidine, clotrimazole, fluconazole, and nystatin) incorporated into a tissue conditioner (Lynal) at zero (control), low, medium, and high concentrations. Hardness of all experimental groups increased with both concentration and time, but remained within the range of clinical acceptability (Shore A < or = 49). Mean tensile strength increased in all experimental groups and was significantly stronger than the control for at least one concentration level of each drug, p < or = 0.05. All failures for tensile-strength testing were characterized as cohesive. All drugs demonstrated release from the tissue conditioner matrix and inhibition of growth of C. albicans that was either total, dose-related, or related to incubation time prior to inoculation. All controls supported growth.