Gas-Phase Fragmentation Analysis of Nitro-Fatty Acids

Nitro-fatty acids are electrophilic signaling mediators formed in increased amounts during inflammation by nitric oxide and nitrite-dependent redox reactions. A more rigorous characterization of endogenously-generated species requires additional understanding of their gas-phase induced fragmentation. Thus, collision induced dissociation (CID) of nitroalkane and nitroalkene groups in fatty acids were studied in the negative ion mode to provide mass spectrometric tools for their structural characterization. Fragmentation of nitroalkanes occurred mainly through loss of the NO₂^- anion or neutral loss of HNO₂. The CID of nitroalkenes proceeds via a more complex cyclization, followed by fragmentation to nitrile and aldehyde products. Gas-phase fragmentation of nitroalkene functional groups with additional γ or δ unsaturation occurred through a multiple step cyclization reaction process, leading to 5 and 6 member ring heterocyclic products and carbon chain fragmentation. Cyclization products were not obtained during nitroalkane fragmentation, highlighting the role of double bond π electrons during NO₂^- rearrangements, stabilization and heterocycle formation. The proposed structures, mechanisms and products of fragmentation are supported by analysis of ¹³C and ¹⁵N labeled parent molecules, 6 different nitroalkene positional isomers, 6 nitroalkane positional isomers, accurate mass determinations at high resolution and quantum mechanics calculations. Multiple key diagnostic ion fragments were obtained through this analysis, allowing for the precise placement of double bonds and sites of fatty acid nitration, thus supporting an ability to predict nitro positions in biological samples.     

Effects of supplemental UV-A on the development, anatomy and metabolite production of Phyllanthus tenellus cultured in vitro

Phyllanthus tenellus is widely used for its antiviral, analgesic and hepatoprotective properties. Although the production of several chemical classes of secondary metabolites is influenced by UV radiation, particularly phenolic compounds, we also know that UV radiation can result in anatomical and developmental damage. However, the morphological, anatomical and phytochemical changes in response to UV-A exposure are generally understudied in the Phyllanthaceae. Therefore, we evaluated the effects of UV-A radiation on plant development and leaf anatomy, as well as the production of secondary metabolites and the contents of carotenoids and chlorophylls a and b, in P. tenellus. To accomplish this, in vitro cultures of P. tenellus were maintained for 60 days under white light (WL) and WL plus UV-A radiation. Results showed different phenotypic responses under additional UV-A, such as high phenolic metabolite production, increasing dimensions of abaxial epidermis and thickness of palisade parenchyma. Compared to plants cultured under WL, UV-A radiation caused damage to plant morphogenesis, including a reduced number of branches and shoots, consequently reducing the rate of proliferation. On the other hand, geraniin, ellagic acid and carotenoid contents increased after UV-A exposure, indicating that this light source is an important resource for inducing phenolic compounds.     

EIS microfluidic chips for flow immunoassay and ultrasensitive cholera toxin detection

A flow-injection impedimetric immunosensor for the sensitive, direct and label-free detection of cholera toxin is reported. A limit of detection smaller than 10 pM was achieved, a value thousands of times lower than the lethal dose. The developed chips fulfil the requirement of low cost and quick reply of the assay and are expected to enable field screening, prompt diagnosis and medical intervention without the need of specialized personnel and expensive equipment, a perspective of special relevance for use in developing countries. Since the chip layout includes two sensing areas each one with a 2 × 2 sensor array, our biochips can allow statistical or (alternatively) multiplex analysis of biorecognition events between antibodies immobilized on each working electrode and different antigens flowing into the chamber.     

Assessment of the in vivo genotoxicity of new lead compounds to treat sickle cell disease

The compounds 1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl nitrate (C1), (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl nitrate (C2), 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitrate (C3), 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-hydroxy-benzenesulfonamide (C4), 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitrate (C5), and 2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate (C6) were evaluated with a micronucleus test using mouse peripheral blood to identify new candidate drugs for the treatment of sickle cell disease (SCD) that are safer than hydroxyurea. The compounds induced an average frequency of micronucleated reticulocytes (MNRET) of less than six per 1,000 cells at 12.5, 25, 50, and 100 mg/kg, whereas hydroxyurea induced an average MNRET frequency of 7.8, 9.8, 15, and 33.7 per 1000 cells respectively, at the same concentrations. Compounds C1-C6 are new non-genotoxic in vivo candidate drugs for the treatment of SCD symptoms.     

Polymorphism, crystallinity and hydrophilic-lipophilic balance of stearic acid and stearic acid-capric/caprylic triglyceride matrices for production of stable nanoparticles

There is an increasing interest in lipid nanoparticles because of their suitability for several administration routes. Thus, it becomes even more relevant the physicochemical characterization of lipid materials with respect to their polymorphism, lipid miscibility and stability, as well as the assessment of the effect of surfactant on the type and structure of these nanoparticles. This work focuses on the physicochemical characterization of lipid matrices composed of pure stearic acid or of mixtures of stearic acid-capric/caprylic triglycerides, for drug delivery. The lipids were analyzed by Differential Scanning Calorimetry (DSC), Wide Angle X-ray Diffraction (WAXD), Polarized Light Microscopy (PLM) and hydrophilic-lipophilic balance (HLB) in combination with selected surfactants to determine the best solid-to-liquid ratio. Based on the results obtained by DSC and WAXD, the selected qualitative and quantitative composition contributed for the production of stable nanoparticles, since the melting and the tempering processes provided important information on the thermodynamic stability of solid lipid matrices. The best HLB value obtained for stearic acid-capric/caprylic triglycerides was 13.8, achieved after combining these lipids with accepted surfactants (trioleate sorbitan and polysorbate 80 in the ratio of 10:90). The proposed combinations were shown useful to obtain a stable emulsion to be used as intermediate form for the production of lipid nanoparticles.     

Rheological Analysis of Biopolymer Produced by Aureobasidium Pullulans in Different Sources of Nitrogen

Summary: In this study, the rheological behaviors of media fermented by two Aureobasidium pullulans strains (IOC 3467 and IOC 3011) were evaluated in different nutritional conditions. The media consisted of crystal sugar (sucrose), as the main carbon source, and different nitrogen sources (ammonium sulfate, sodium nitrate, ammonium nitrate, urea or residual brewery yeast - RBY). Viscosity measurements were performed on cell-free supernatants, from 48-hour fermentation assays, at 25 ^°C. Shear rates ranged between 0.1000 and 500 s⁻¹. All samples showed pseudoplastic behavior. Nevertheless, the viscosimetric profile of each one varied according to the nitrogen source, its concentration and the strain used. The maximum viscosity of 0.06 Pa.s. was achieved at 15.6 s⁻¹ for IOC 3011 strain grown on RBY as nitrogen source.     

Analysis of phase transition and dehydration processes of nevirapine

Solid-state characterization of crystalline drugs is an important pre-formulation step for the development and design of solid dosage forms, such as pellets and tablets. In this study, phase transition and dehydration processes of nevirapine have been studied by differential scanning calorimetry and thermogravimetry differential thermal analysis to overcome the problems of drug formulation, namely poor solubility and poor content uniformity. Phase solubility studies elucidated the mechanism of enhanced nevirapine solubility.     

Applied biological and physicochemical activity of isoquinoline alkaloids: oxoisoaporphine and boldine

The aim of this study was to determine the electronic influence of substituent groups and annelated rings such as oxazole-oxazinone on the physicochemical and photoprotection, antioxidant capacity, toxicity and singlet oxygen photosensitization biological properties of isoquinoline alkaloid frameworks. Thus, oxoisoaporphine derivatives 1-5 and 3-azaoxoisoaporphine (6), some of them with phenolic structures, did not present any antioxidant capacity, possibly either by formation of keto-enol tautomerism species or the formation of unstable free radicals. Due to the singlet oxygen quantum yields (F_D) near to unity, and greater photostability than phenalenone, oxoisoaporphines 4-6 may be considered as photosensitizers for singlet oxygen production and can be used as new universal study tools. The biological application as antibacterial agents is an important and possible tool in the study of compounds with low cytotoxicity and high reactivity in antineoplastic chemotherapy. On the other hand, when boldine and its annelated derivatives B1-4 are irradiated, a photoprotector effect is observed (SPF = 2.35), even after 30 minutes of irradiation. They also act as photoprotectors in cell fibroblast cultures. No hemolysis was detected for boldine hydrochloride and its salts without irradiation. In solutions irradiated before incubation (at concentrations over 200 ppm) photoproducts were toxic to the nauplii of Artemia salina.     

Chalcones in bioactive Argentine propolis collected in arid environments

The aim of this study was to assess the chemical and biological profile of propolis samples collected in arid environments of north-western Argentina. The samples were from two phytogeographical regions (Prepuna and Monte de Catamarca Province). Propolis ethanolic extracts (PEE) and chloroform (CHL), hexane (HEX) and aqueous (AQ) sub-extracts of samples from three regions (CAT-I; CAT-II and CAT-III) were obtained. All PEE exhibited antioxidant activity in the DPPH radical scavenging assay (SC50 values between 28 and 43 microg DW/mL). The CHL extract was the most active (SC50 values between 10 and 37 microg DW/mL). The antioxidant activity in the beta-carotene bleaching assays was more effective for PEE and CHL (IC50 values between 2 and 9 microg DW/mL, respectively). A similar pattern was observed for antibacterial activity. The highest inhibitory effect on the growth of human Gram-positive bacteria was observed for CHL-III and CHL-I (Monte region) with minimal inhibitory concentration values (MIC100) of 50 to 100 microg DW/mL. Nine compounds were identified by HPLC-PAD. Two of them (2', 4'- dihydroxychalcone and 2',4'- dihydroxy 3'-methoxychalcone) were found only in propolis samples from the Monte phytogeographical region. We consider that the Argentine arid region is appropriate to place hives in order to obtain propolis of excellent quality because the dominant life forms in that environment are shrubby species that produce resinous exudates with a high content of chalcones, flavones and flavonols.     

Reduced Fluoresceinamine for Peroxynitrite Quantification in the Presence of Nitric Oxide

A new fluorescent analytical methodology for the quantification of peroxynitrite (ONOO(-)) in the presence of nitric oxide (NO) was developed. The quantification of ONOO(-) is based in the oxidation of the non-fluorescent reduced fluoresceinamine to a high fluorescent oxidized fluoresceinamine in reaction conditions where the interference of NO is minimized. Screening factorial experimental designs and optimization Box-Behnken experimental design methodologies were used in order to optimize the detection of ONOO(-) in the presence of NO. The factors analysed were: reduced fluoresceinamine concentration (C( Fl)); cobalt chloride concentration (C(CoCl2)); presence of oxygen (O(2)); and, the pH (pH). The concentration of sodium hydroxide (C(NaOH)) needed to diluted the initially solution of ONOO(-) was also evaluated. An optimum region for ONOO(-) quantification where the influence of NO is minimal was identified - C(Fl) from 0.50 to 1.56 mM, C(CoCl2) from 0 to 1.252?×?10(-2) M, pH from 6 to 8 and C(NaOH) 0.10 M. Better results were found in the presence of NO at pH 7.4, C(Fl) 0.5 mM, without oxygen, without cobalt chloride and with a previous dilution of peroxynitrite solution with C(NaOH) 0.1 M. This methodology shows a linear range from 0.25 to 40 μM with a limit of detection of 0.08 μM. The bioanalytical methodology was successfully applied in the ONOO(-) quantification of fortified serum and macrophage samples.