Exposure of human skin to benzo[a]pyrene: role of CYP1A1 and aryl hydrocarbon receptor in oxidative stress generation.

Exposure to benzo[a]pyrene (BaP) can induce inflammatory skin diseases and skin cancer, which are both associated to oxidative stress. BaP is known to bind with high specificity to the aryl hydrocarbon receptor (AhR), modifying the expression of CYP1A1, involved both in cancer and inflammation. While the current knowledge is based on murine skin and cell culture data, in this study human healthy skin has been treated with 5muM BaP in conditions simulating occupational and environmental exposure. AhR and CYP1A1 expression was evaluated by Western blotting, which revealed their presence even in control untreated skin; both enzyme and receptor increased more than twofold after exposure to BaP. AhR expression level was lower than CYP1A1 in basal conditions and following induction. Oxidative stress was evaluated in terms of MTT reduction, protein peroxidation and reactive oxygen species (ROS) formation. A significant increase in ROS and carbonyl compound production, as well as reduced tissue viability have been determined by BaP. The results of this experiment indicate that BaP, an AhR agonist, can significantly increase receptor and CYP1A1 expression and induce oxidative stress in human skin, confirming the involvement of this pathway in the pathogenesis of tissue damage due to polycyclic aromatic hydrocarbons.

Physico-chemical characterization of an amphiphilic cyclodextrin/genistein complex.

Specific recognition of cell-targeting systems as host-carriers modified with receptor targeting groups, is a major ambition in the application of supramolecular science to medicine and life science. Genistein (Gen), an isoflavone belonging to the class of phytoestrogens, is of great interest because it has been considered as potential remedy for many kinds of disease. In this work, genistein in aqueous medium and in the presence of an host nanocarrier as amphiphilic cyclodextrin (CyD) modified in the upper rim with oligoethylene hydroxyl groups [(2-oligo(ethyleneoxide)-6-hexylthio)-beta-CyD, SC6OH] at 1:1 molar ratio, has been firstly investigated by UV-vis measurements coupled with circular dichroism data, in order to characterize the drug/macrocycle binding affinity through the formation of the complex. Furthermore, FTIR-ATR technique has been used to detect the complex formation in solid phase and to characterize the functional groups responsible of the solid Gen/SC6OH complex stability. The infrared absorbance spectra of the complex, collected in a wide range of wavenumber and around the physiological temperature, have been analysed and compared with the spectra of the pure compounds and their physical mixture. By monitoring the most significant changes in the shape and position of the absorbance bands of the Gen functional groups, we showed that the formation and/or modification of polar bonds play the main role in the interaction of the drug with the amphiphilic CyD. From the results, Gen is shown to be entangled in SC6OH nanoaggregates, establishing hydrogen bonding with the hydrophilic PEG chains.

UV-vis and FTIR-ATR characterization of 9-fluorenon-2-carboxyester/(2-hydroxypropyl)-beta-cyclodextrin inclusion complex.

In this work, the usefulness of (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CyD) as a tool to form an inclusion complex with 9-fluorenonic derivative (AG11) has been investigated, in pure water, by UV absorption. Phase-solubility diagrams allowed the determination of the association constant between AG11 and HP-beta-CyD. At the same time, solid binary systems between AG11 and HP-beta-CyD have been prepared in 1:1 stoichiometry by co-precipitation method. In order to confirm the complexation, FTIR spectroscopy in ATR geometry measurements have been performed and the results have been compared with the free compounds and the corresponding physical mixture in the same molar ratio. The nature of the interactions between AG11 and HP-beta-CyD has been elucidated also by applying mathematical procedures such as deconvolution and curve fitting. Improvement of the aqueous solubility is expected to improve the bioavailability of the drug in oral administration.

The enhancement of isoflavones water solubility by complexation with modified cyclodextrins: a spectroscopic investigation with implications in the pharmaceutical analysis.

The improvement of isoflavones bioavailability by complexation with chemically modified cyclodextrins (CyDs) has been exploited to analyse the drug/macrocycle binding affinity by a conventional method with new useful measures. Genistein (Gen) and daidzein (Daidz) were investigated in aqueous medium and in presence an amount of (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CyD) at different host/guest molar ratios. The solubility in pure water, approximately 3 x 10(-6)M for Gen and approximately 10 x 10(-6)M for Daidz, was obtained by distributing the of guest molecule between water and the organic solvent. The stoichiometric ratios and stability constants describing the extent of formation of the complexes have been determined by phase-solubility UV-vis measurements and confirmed by circular dichroism data. These results have implications in the determination of the carrier's capacity for the complexation of the drug in water solution.

UV-vis and FTIR-ATR spectroscopic techniques to study the inclusion complexes of genistein with beta-cyclodextrins.

The effect of beta-cyclodextrin (beta-CyD), (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CyD) and methyl-beta-cyclodextrin (Me-beta-CyD) complexation on the UV absorption of genistein (Gen) was studied in pure water. A phase solubility study was performed, according to the method reported by Higuchi and Connors, to evaluate the changes of isoflavone in the complexation state and the obtained diagrams suggested that it forms complexes with a stoichiometry of 1:1. Then, the solid complexes of genistein with these macrocycles in 1:1 molar ratio were prepared by the co-precipitation method and characterized by FTIR absorption spectroscopy in ATR geometry. The host-guest interactions have been evidenced by monitoring, in the FTIR-ATR spectra, the changes in some guest molecule bands relative to those observed in the spectra of the 1:1 physical mixtures and complexes. In particular, for the high-frequency O-H stretching band, a quantitative vibrational assignment of the observed sub-bands has been made. From the results, the inclusion phenomena have been discussed.

Optimization of a LC method for the enantioseparation of a non-competitive glutamate receptor antagonist, by experimental design methodology.

The aim of this work was to obtain the direct optical resolution of a new glutamate receptor antagonist ((p-chloro)1-aryl-6,7,-dimethoxy-1,2,3,4-tetrahydroisoquinoline, PS3), by liquid chromatography on Chiralcel OD column. A response surface methodology (RSM) was employed to optimize the enantiomeric separation of the racemate with the lowest number of experiments; in particular, a face-centred design (FCD) was applied to evaluate the influence of critical parameters on the experimental response. Furthermore, in order to find the best compromise between several responses, a multicriteria decision-making approach, the Derringer's desirability function, was successful to simultaneously optimize the responses resolution and migration times of the two enantiomers. The proposed LC method provided the baseline enantioseparation of the investigated drug. 9.3% (v/v) ethanol added to n-hexane as mobile phase, 1.0 mL min(-1) flow rate, and 18 degrees C column temperature were the optimum experimental conditions allowing to achieve the highest enantioresolution of PS3 in less than 17 min.

The inclusion complexes of hesperetin and its 7-rhamnoglucoside with (2-hydroxypropyl)-beta-cyclodextrin.

The effect of (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CyD) on the solubility properties and spectroscopic features of hesperetin and its 7-rhamnoglucoside, hesperidin, was qualitatively and quantitatively investigated in water, by means of UV-vis absorption and fluorescence spectroscopy. The stoichiometric ratios and stability constants describing the extent of formation of the complexes have been determined by phase-solubility measurements; in both cases type-A(L) diagrams have been obtained (soluble 1:1 complexes). The higher degree of interaction showed by hesperetin may be attributed to the higher hydrophobicity and smaller size of the aglycone molecule, which therefore exhibits a greater affinity for the CyD and fits better into the cavity. The effect of molecular encapsulation on the two flavanones antioxidant activity was afterwards evaluated by means of different biological assays, concerned to the different mechanisms of in vivo action. The protection efficacy was in all cases higher for the complexed drugs, with respect to the free ones; these results are of great interest for their potential usefulness in pharmaceutics.

The rutin/beta-cyclodextrin interactions in fully aqueous solution: spectroscopic studies and biological assays.

In the present work the feasibility of beta-cyclodextrin complexation was explored, as a tool for improving the aqueous solubility and antioxidant efficacy of rutin. By means of 1H NMR, UV-vis and circular dichroism spectroscopy the single aromatic ring of rutin was found to be inserted into the beta-cyclodextrin cavity to form a 1:1 inclusion complex. The effect of beta-cyclodextrin on the spectral features of rutin was quantitatively investigated, in fully aqueous medium, by holding the concentration of the guest constant and varying the host concentration. The associated binding constants were estimated to be 142+/-20 and 153+/-20 M(-1), respectively, on the basis of the observed UV-vis absorption and circular dichroism intensities. The antioxidant activity of rutin was also investigated, as affected by molecular encapsulation within beta-cyclodextrin (batophenanthroline test; comet assay; lipid peroxidation); the inclusion complex revealed improved antioxidant efficacy that may be in part explained by an increased solubility in the biological moiety.

Effects of alpha- and beta-cyclodextrin complexation on the physico-chemical properties and antioxidant activity of some 3-hydroxyflavones.

Inclusion complexes of some flavonols (3-hydroxyflavone, morin and quercetin) have been obtained with alpha- and beta-cyclodextrins, by the co-evaporation method. Different analytical techniques (DSC, XRPD, FT-IR, 1H-NMR, UV-Vis) have been employed for a throughout investigation of the structural characteristics of such supramolecular aggregates, which exhibited distinct spectroscopic features and properties from both "guest" and "host" molecules. The stoichiometric ratios and stability constants describing the extent of formation of the complexes have been determined by phase-solubility studies; in all cases type-AL diagrams have been obtained (soluble 1:1 complexes). The effect of molecular encapsulation on the flavonols antioxidant activity has been afterwards evaluated, by means of different biological assays (Bathophenanthroline test; Comet assay; Lipid peroxidation). Complexation with cyclodextrins further improved the antioxidant activity, increasing drugs solubility in the biological moiety.

LC-MS for the identification of oxygen heterocyclic compounds in citrus essential oils.

The oxygen heterocyclic compounds (coumarins, psoralens and polymethoxylated flavones) present in the nonvolatile residue of the essential oils of Mandarin, Sweet Orange, Bitter Orange, Bergamot and Grapefruit were analysed with an HPLC/API/MS system equipped with an APcI probe in positive mode. The use of hyphenated techniques, such as LC/MS provides a great information about the content and nature of constituents of natural complex matrices, such as essential oils. In this work, MS spectra were recorded at different voltages, to obtain structural information in addition to molecular weight information. The different response of the compounds identified has been also evaluated. The method allowed the confirmation of the identification of the main components of the fraction, previously reported for the different oils. MS characteristics of coumarins, psoralens and polymethoxylated flavones with different substitution patterns were determined on the basis of the response obtained with the APcI interface. Interface parameters were optimised to obtain a contemporaneous response for all the three classes of components.

Rapid analysis of essential and branched-chain amino acids in nutraceutical products by micellar electrokinetic capillary chromatography.

A rapid method for the analysis of dansylated essential and branched-chain amino acids (BCAAs) by micellar electrokinetic capillary chromatography (MECC) is reported. Optimization of analytical conditions has been carried out, evaluating the influence on the performance of several parameters such as sodium dodecyl sulfate (SDS) concentration in the running electrolyte, temperature, and voltage. The effect of the addition of small amounts of isobutanol to the electrolyte has also been investigated. The best separation in the shortest time with a 37 cm capillary was obtained employing a 20 mM Borax buffer (pH 9.1) + 70 mM SDS at 25 degrees C and 20 kV. Under these conditions a mixture of nine essential amino acids was analyzed in 7 min, while separation of BCAAs occurred in less than 4 min. Using a shorter capillary (20 cm to the detector), the BCAA separation was performed in only 2.5 min. The method was applied to the quantitative analysis of amino acids in three commercial nutraceutical preparations. Assessment of analytical performance in terms of precision, linearity, and limit of detection has also been reported.

Stability study of piroxicam and cinnoxicam in solid pharmaceuticals.

The pseudo-first order rate constant for the hydrolysis of cinnoxicam as a function of temperature was obtained by variable-temperature kinetic experiments. The method used is on a generalization of non-isothermal analysis, and takes advantage of the capabilities of modern data collection and processing systems. A spectrophotometric method under non isothermal conditions was carried out. The results obtained are identical to those obtained under the same conditions by using traditional constant-temperature kinetic runs. This provides the possibility of reducing the amount of time spent and chemicals usually used in collecting kinetic data in mechanistic studies in solution by an order of magnitude.