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.

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.

Combined effect of pH and polysorbates with cyclodextrins on solubilization of naringenin.

pH control and inclusion complex formation are commonly used as solubilization techniques in formulating ionizable drugs. Naringenin is a weakly acid compound with a low water solubility. The role of both ionized and unionized species of naringenin in solution by complexation with beta-cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin and methyl-beta-cyclodextrin was investigated. This combined use of ionization and complexation increases not only the solubility of the unionized naringenin, but also that of the ionized one. This study puts on evidence the role of pH, pKa and complexation constants in increasing drug total aqueous solubility, determined by the single components in solution, as ionized and unionized naringenin both in free and complexed forms. Moreover, the presence of non-ionic surfactants in the media of complexation gives a positive contribution to the improvement of the solubility of naringenin, alone or in combination with beta-cyclodextrin.

Study of the extraction procedure by experimental design and validation of a LC method for determination of flavonoids in Citrus bergamia juice.

A reversed-phase high-performance liquid chromatographic (HPLC) separation with photo-diode array detection was developed for the simultaneous determination of flavonoids extracted from Citrus bergamia juice. It employs a C18 reversed-phase column and a linear gradient elution system with methanol/water with 5% acetic acid (v/v), as mobile phase. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, precision and accuracy. Limits of detection ranged from a low of 0.007 mg ml(-1) (narirutin) to a high of 0.018 mg ml(-1) (didymin). The limits of quantitation were between a low of 0.011 mg ml(-1) (7-OH flavanone) and a high of 0.024 mg ml(-1) (didymin). An excellent linear response was observed over the range specified for all analytes, as confirmed by the correlation coefficient with ranged from 0.9982 and 0.9999. The intra-day R.S.D.% ranged from 0.11 to 3.64%. The intermediate precision R.S.D.% were not higher than 7.62%. The accuracy of the method was confirmed with an average recovery ranging, except for neoeriocitrin, between 88.07% and 102.45%. Since the extraction conditions can affect analyte recovery, a suitable optimization strategy of the procedure was needed. The experimental parameters optimized were extraction time, temperature, and solvents. A multivariate approach was used to provide direct evaluation of the selected variables and related interactions. The D-optimal design was constructed by applying the exchange algorithm. All experimental results were computed by NEMROD-W software. This methodology led us to obtain the best recovery for all the flavonoids in the least number of experiments.

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.

Comparative photodegradation studies on 3-hydroxyflavone: influence of different media, pH and light sources.

3-Hydroxyflavone (3-OH-F) photochemistry in solution has been rationalized in terms of an excited state intramolecular proton transfer (ESIPT), which involves the free 3-hydroxy group interacting with the ortho-carbonyl. This photo-rearrangement occurs rapidly and is strongly influenced by the physico-chemical properties of the solvent, which plays an essential role in determining whether a photo-oxidation or a photo-induced molecular rearrangement takes place. 3-OH-F photoreactivity has been deeply investigated and the related mechanisms elucidated, as affected by various solvents, pH values and irradiation wavelengths, leading to different photodegradation rates and pathways. Moreover, the influence of molecular encapsulation upon alpha- and beta-cyclodextrins (alpha- and beta-CyD) on the molecule photoreactivity has been examined, as a potential tool for increasing molecule photostability as well as minimizing photoinduced toxic effects on biosubstrates.

Selective reversed-phase liquid chromatography method for the kinetic investigation of 3-hydroxyflavone photostability.

This paper reports a fast and accurate RP-HPLC chromatographic method for the simultaneous determination of 3-hydroxyflavone (3-OH F) and its photodegradation products. Solutions (5 x 10(-5) M) in acetonitrile (ACN) of the molecule were subjected to forced degradation by exposure to artificial UV-A light source (black-light, lambda(max) 354 nm) and the changes appearing in chromatograms were monitored at selected irradiation times. A multistep gradient was optimised to achieve complete elution of all photoproducts in the shortest analysis time. UV spectra recorded by the diode array detector system (285 and 340 nm) clearly showed the structural changes in the new species formed, with respect to the parent compound. The analytical method was subjected to a validation procedure in which linearity and range, as well as specificity, precision and accuracy were determined according to ICH guidelines. Quantitative evaluation of the photochemical process was performed on the basis of the calculated kinetic parameters: photodegradation rate constant k, half-life time t(0.5), time degradation of 10% of the drug t(0.1).

An experimental design methodology applied to the enantioseparation of a non-steroidal anti-inflammatory drug candidate.

An experimental design methodology has been applied to the enantioseparation of a new synthesized aryl propionic acid of pharmaceutical interest, namely 2-[(4'-benzoyloxy-2'-hydroxy)phenyl-propionic acid] (DF-1770y) by chiral capillary zone electrophoresis (CCZE). The chiral separation of the studied compound has been achieved employing vancomycin as the chiral selector. The partial filling-counter current method has been used in order to avoid the presence of the absorbing chiral selector in the path length of the detector and to increase the method sensitivity. A central composite design has been employed to optimize the experimental conditions for a fast separation of the enantiomers of the new synthesized aryl propionic acid. Critical parameters such as chiral selector concentration, pH and temperature have been studied to evaluate how they affected responses such as resolution and migration times. The desirability function approach has been employed in order to find the best compromise between the different experimental responses. The proposed CCZE method provided the baseline enantioseparation of the investigated drug. A Britton-Robinson buffer at pH 6.4 supplemented with 7 mM of vancomycin at 22 degrees C and -20 kV were the optimum experimental conditions allowing to achieve the highest enantioresolution of DF-1770y in less than 8.5 min.

Study of flavonoids/beta-cyclodextrins inclusion complexes by NMR, FT-IR, DSC, X-ray investigation.

Flavonoids are natural substances with a lot of biological activities, including the antioxidant one. Their use in pharmaceutical field is, however, limited by their aqueous insolubility. As the formation of the inclusion complexes can improve their solubility in water, the flavonoids hesperetin, hesperidin, naringenin and naringin have been complexed with beta-cyclodextrin (beta-CD) by the coprecipitation method and studied in solution and in solid state by NMR, FT-IR, differential scanning calorimetry and X-ray techniques. The effects of complexation on the chemical shifts of the internal and external protons of beta-CD in the presence of each flavonoid were observed.

Validation of a LC method for the analysis of oxaliplatin in a pharmaceutical formulation using an experimental design.

A rapid and sensitive RP-HPLC method with UV detection for routine control of oxaliplatin in a pharmaceutical formulation (Eloxatin) was developed. Quantitation was accomplished with the internal standard method. The procedure was validated by linearity (correlation coefficient=0.999948), accuracy, robustness and intermediate precision. Experimental design was used during validation to calculate method robustness and intermediate precision. For robustness test three factors were considered: percentage v/v of acetonitrile, flow rate and temperature; an increase in the flow rate results in a decrease of the drug found concentration, while the percentage of organic modifier and temperature have no important effect on the response. For intermediate precision measure the considered variables were: analyst, equipment and days. The RSD value (2.27%, n=24) indicated a good precision of the analytical method.

Study of beta-blockers/beta-cyclodextrins inclusion complex by NMR, DSC, X-ray and SEM investigation.

The formation of inclusion complexes between beta-cyclodextrin with the two beta-blockers, atenolol and celiprolol, have been studied in the aqueous environment and in the solid state by nuclear magnetic resonance (NMR) spectroscopy, X-ray, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) techniques. The magnitude of the chemical shifts of the interior and exterior beta-cyclodextrin protons in the presence of each beta-blocker indicated that these are included within the beta-cyclodextrin cavity. In aqueous solution they form 1:1 complexes. In the solid state the formation of the beta-cyclodextrin/atenolol (celiprolol) complexes is confirmed by X-ray, DSC and SEM, also employed to characterize pure substances and their physical mixtures.

Validation of a LC method for the analysis of zafirlukast in a pharmaceutical formulation.

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed and validated for estimation of zafirlukast in a pharmaceutical formulation. Assay samples were extracted utilizing acetonitrile. Drug and internal standard were chromatographed on reversed-phase C18 columns, using mixtures of acetonitrile/water and the eluents were monitored at different wavelengths. The method was validated statistically for its linearity, accuracy, robustness and precision. Experimental design was used during validation to evaluate method robustness and for the determination of intermediate precision. Factors examined for statistical approaches include laboratory, day, analyst, instrument, different percentage of organic modifier, temperature, wavelength and flow-rate. Due to its simplicity and accuracy, the method may be used for routine quality control analysis.

Isolation and characterization of Guiera senegalensis J.F.Gmel. active principles.

A combination of thin-layer chromatography, high-performance liquid chromatography, UV spectroscopy and NMR spectrometry techniques for analysis of flavonoid content of Guiera senegalensis J.F.Gmel. (Combretaceae) leaves is described. Four components, catechin, myricitrin, rutin and quercetin were evidenced and determined by HPLC.

Direct enantiomeric separation of flavanones by high performance liquid chromatography using various chiral stationary phases.

The performance of four chiral liquid chromatographic columns (Chiralcel OA, OJ, OC, OD) was studied with respect to the enantioseparation of flavanones and some derivatives. The effect of mobile phase composition on the retention time and stereo-selectivity was studied. A good enantioseparation (alpha up to 1.45) was achieved for most of the racemates. Further, the elution order of the enantiomers from the chiral stationary phases has been determined by using a circular dichroism based detection system.