Coumarin/ß-Cyclodextrin Inclusion Complexes Promote Acceleration and Improvement of Wound Healing.

Coumarins have great pharmacotherapeutic potential, presenting several biological and pharmaceutical applications, like antibiotic, fungicidal, anti-inflammatory, anticancer, anti-HIV, and healing activities, among others. These molecules are practically insoluble in water, and for biological applications, it became necessary to complex them with cyclodextrins (CDs), which influence their bioavailability in the target organism. In this work, we studied two coumarins, and it was possible to conclude that there were structural differences between 4,7-dimethyl-2H-chromen-2-one (DMC) and 7-methoxy-4-methyl-2H-chromen-2-one (MMC)/ß-CD that were solubilized in ethanol, frozen, and lyophilized (FL) and the mechanical mixtures (MM). In addition, the inclusion complex formation improved the solubility of DMC and MMC in an aqueous medium. According to the data, the inclusion complexes were formed and are more stable at a molar ratio of 2:1 coumarin/ß-CD, and hydrogen bonds along with π-π stacking interactions are responsible for the better stability, especially for (MMC)2@ß-CD. In vivo wound healing studies in mice showed faster re-epithelialization and the best deposition of collagen with the (DMC)2@ß-CD (FL) and (MMC)2@ß-CD (FL) inclusion complexes, demonstrating clearly that they have potential in wound repair. Therefore, (DMC)2@ß-CD (FL) deserves great attention because it presented excellent results, reducing the granulation tissue and mast cell density and improving collagen remodeling. Finally, the protein binding studies suggested that the anti-inflammatory activities might exert their biological function through the inhibition of MEK, providing the possibility of development of new MEK inhibitors.

Magnetic and mesoporous molecularly imprinted polymer synthesized by rational computation design: Sample preparation and analysis of ractopamine.

Through density functional theory calculations was studied theoretically the formation process of a magnetic and mesoporous molecularly imprinted polymer for ractopamine (RAC), evaluating the molecular electrostatic potential map, functional monomers, functional monomer / template stoichiometry and crosslink agents. The results revealed that the best conditions for the synthesis were established with acrylic acid as functional monomer in a 1: 4 stoichiometry using acetonitrile as the solvent and ethylene glycol dimethacrylate as crosslink agent. It was observed that nine hydrogen bonds established between the RAC and acrylic acid play a key role on the pre-polymerization complex. In addition, three analytical methods using HPLC, UHPLC and CE instruments were optimized for rapid analysis. The adsorbent was experimentally synthesized considering the best conditions found at the molecular level and characterized by FTIR, DRX, TGA, SEM, TEM, surface analysis, and wettability. After that, the synthesized material was used in magnetic solid phase extraction combined with capillary electrophoresis in a preliminary RAC recovery study from milk samples. Finally, greenness metric with a score of 0.55 have been obtained for the sample preparation procedure using the online AGREEprep metric.

Restricted access polypyrrole employed in pipette-tip solid-phase extraction for determination of nimodipine and nicardipine in breast milk.

A HPLC-UV method for the determination of nimodipine and nicardipine in breast milk using restricted access polypyrrole as an adsorbent in pipette-tip solid-phase extraction (PT-SPE) has been developed. The chromatographic conditions were a C18 column (150 mm × 4.60 mm, 5 µm) using methanol : acetonitrile : ultrapure water (55 : 30 : 15, v/v/v) at a flow rate of 1.0 mL min-1 and detection at 236 nm. The adsorbents have been synthesized and characterized by using Fourier-transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, surface analysis, wettability and point zero charge, and were then applied in sample preparation. The main parameters that affect analyte recovery from breast milk by PT-SPE were optimized and the analytical method showed recoveries around 100%, linearity from 3 to 3000 ng mL-1, and correlation coefficients (r) ≥ 0.99 for the two analytes, in addition to adequate precision, accuracy and robustness. Finally, the validated method has been successfully applied in analyses of breast milk from volunteers.

Magnetic molecularly imprinted conducting polymer for determination of praziquantel enantiomers in milk.

A new selective adsorbent based on magnetic molecularly imprinted conducting polymer was firstly synthetized and applied to the magnetic solid phase extraction (MSPE) for the determination of PZQ enantiomers in milk samples. The enantioselective separation was performed on a Chiralpak® IB column using mobile phase composed of ultrapure water : tetrahydrofuran : diethylamine (65 : 35 : 0.05, v/v/v). After MSPE optimization, in which a magnetic molecularly imprinted polypyrrole (MMIPPy) was used as adsorbent, the best conditions were: 500 µL of acetonitrile as eluent, 75 mg of MMPPy, 1000 µL of milk sample (pH adjusted to 6.5.). The analyses showed recoveries/relative standard deviation (RSD%) 79.7 ± 2.5 and 81.1 ± 2.2 for (R)-(-)-PZQ and (S)-(+)-PZQ, respectively. By computational study carried out on the molecular design of the synthesized MMIPPy, it was found that the formation of four hydrogen bonds and one π-π stacking interaction explain the formation of the PZQ-pyrrole pre-polymerization complex at the molecular level. From the analytical validation, the developed method showed to be linear in the concentration range of 0.01 to 10 µg mL-1, with correlation coefficient larger than 0.98 and RSD% values below 15%. The LOQ obtained was 0.01 µg mL-1 for both enantiomers, with RSD% and relative error below 20%. The method was successfully applied in real sheep milk samples from various local suppliers. Finally, the results showed that MMIPPy in MSPE is an economical, simple and easy-to-perform technique.

Elucidation of the chromatographic enantiomer elution order for praziquantel: An experimental and theoretical assessment.

In this work, we have studied both experimentally and theoretically the praziquantel (PZQ) chiral discrimination. According to the main results, the enantioseparation of PZQ was efficiently optimized by HPLC on the reverse phase from the Chiralpak IB column, which has cellulose tris (3,5-dimethylphenylcarbamate) (CDMPC) as a chiral selector. The thermodynamic and structural parameters obtained via density functional theory (DFT) calculations pointed out the chiral discrimination as well as the enantiomeric elution order of PZQ, thus elucidating the experimental data and validating our proposed method. Finally, the hydrogen bonds and π-π stacking interactions played a key role in the discrimination between the PZQ diastereomeric complexes formed.

Development and validation of an experimental and theoretical method for the chiral discrimination of dinotefuran.

Dinotefuran is a low-cost agrochemical considered a highly toxic product. In this sense, there is a need for its constant environmental, biological, and food control, aiming to ensure its use to humans as well as to preserve biodiversity and ecosystems. In the present work, we developed an experimental and theoretical method for dinotefuran chiral discrimination. According to the main results, the dinotefuran enantioselective separation was efficiently optimized by high-performance liquid chromatography evaluating the influence of different percentage compositions in the mobile phase to improve the resolution of the peaks in the chromatogram. The novelty of this work was the proposition of a reduced molecular model for the chiral selector amylose-Tris-(3,5-dimethylphenylcarbamate) polysaccharide that was able to adequately describe at the molecular level its interaction with the dinotefuran enantiomers. Besides, the thermodynamic and structural parameters obtained via density functional theory calculations pointed out the chiral discrimination as well as the enantiomeric elution order of the analyte studied, confirming the experimental data, thus validating our proposed method. Finally, hydrogen bonds and repulsive interactions played a key role in the discrimination between the diastereomeric complexes, and consequently, for the dinotefuran enantioselective separation.

Rational design of a molecularly imprinted polymer for dinotefuran: theoretical and experimental studies aimed at the development of an efficient adsorbent for microextraction by packed sorbent.

In this work, we studied theoretically the formation process of a molecularly imprinted polymer (MIP) for dinotefuran (DNF), testing distinct functional monomers (FM) in various solvents through density functional theory calculations. The results revealed that the best conditions for MIP synthesis were established with methacrylic acid (MAA) as FM in a 1 : 4 stoichiometry and with chloroform as the solvent. This protocol showed the most favourable stabilization energies for the pre-polymerization complexes. Furthermore, the formation of the FM/template complex is enthalpy driven and the occurrence of hydrogen bonds between the DNF and MAA plays a major role in the complex stability. To confirm the theoretical results, MIP was experimentally synthesized considering the best conditions found at the molecular level and characterized by scanning electron microscopy and thermogravimetric analysis. After that, the synthesized material was efficiently employed in microextraction by packed sorbent combined with high-performance liquid chromatography in a preliminary study of the recovery of DNF from water and artificial saliva samples.

Efficient molecularly imprinted polymer as a pipette-tip solid-phase sorbent for determination of carvedilol enantiomers in human urine.

In this work, an efficient pipette tip based on molecularly imprinted polymers solid-phase extraction (PT-MIP-SPE) method was developed for carvedilol (CAR) analysis. This compound is available in clinical practice as a racemic mixture, in which (-)-(S)-CAR is a ß- and α1-adrenergic antagonist, while (+)-(R)-CAR only acts as an α1-adrenergic antagonist. Enantioseparation of CAR presented satisfactory retention times (5.85 and 14.84min), acceptable theoretical plates (N=2048 and 2018) and good resolution (Rs=9.27). The separation was performed using a Chiralpak® IA column (100mm×4.6mm, 3µm), a mixture of methanol:ethanol:water (64:15:21, v/v/v) plus 0.3% diethylamine as mobile phase, temperature of 35°C and flow rate of 1.5mLmin-1. After density functional theory calculations based on prepolymerization complexes, the best protocol for the MIP synthesis was chosen. Then, some parameters that affect the PT-MIP-SPE technique were investigated. After optimization, the best conditions were 300µL of water as washing solvent, 500µL of acetonitrile:acetic acid (7:3, v/v) as eluting solvent, 20mg of MIP, 500µL of urine sample (pH 12.5) and no addition of NaCl. Recoveries±relative standard deviation (RSD%) for (+)-(R)-CAR and (-)-(S)-CAR were 101.9±4.8% and 104.6±2.1%, respectively. The method was linear over the concentration range from 20 to 1280ngmL-1 for each enantiomer, with correlation coefficients larger than 0.99 for both enantiomers. The method was applied successfully in a preliminary study of urinary excretion after administration of CAR racemate to a healthy volunteer.

Computational contribution to the electrophoretic enantiomer separation mechanism and migration order using modified ß-cyclodextrins.

Capillary electrophoresis (CE) is an extremely effective technique in many kinds of separations, including separation of enantiomers. Some additional techniques may be necessary to determine the enantiomer migration order (EMO) and also the mechanism involved in chiral recognition. This paper reports the development and optimization of a CE method for enantioseparation of racemic mixture of both R- and S-stereoisomers of tramadol (TRM) with a computational contribution for the EMO determination and the responsible mechanisms for chiral distinction. Parameters such as composition and concentration of background electrolyte (BGE) and type and concentration of cyclodextrins (CD) were evaluated. For calculations, a sequential methodology was used, resorting to semiempirical Parametric Model 3 (PM3) followed by calculations accomplished using density functional theory. The best results were obtained with sulfated-ß-CD (s-ß-CD) and carboxymethyl-ß-cyclodextrin (cm-ß-CD) as chiral selector. Calculations show that the inclusion of TRM is not a probable process due to the shape of the TRM molecule and the size CDs cavities. Therefore, the chiral recognition process occurs by the formation of association complexes between modified ß-CD and groups of TRM molecules. The structural analysis of the fragments of complexes at a pH of 10 and a thermodynamic analysis of the complexes' formation process allows determining the EMO. Comparing results obtained experimentally and computationally, it seems that the developed method is adequate for separation of TRM enantiomers and the computational methodology is also adequate to get a sense of the system at a molecular level.

Molecular modeling study of the recognition mechanism and enantioseparation of 4-hydroxypropranolol by capillary electrophoresis using carboxymethyl-ß-cyclodextrin as the chiral selector.

The purpose of this paper was to study at the molecular level the enantioseparation mechanism of 4-hydroxypropranolol (4-OH-Prop) with carboxymethyl-ß-cyclodextrin (CM-ß-CD) using a sequential methodology which included molecular dynamics simulations (MD), and Parametric Model 3 (PM3) semiempirical and density functional theory (DFT) calculations. A systematic structural analysis indicated that hydrogen bonds formed between the host and guests play a major role in the complex stabilization. The inclusion complex (+)-(R)-4-OH-Prop/CM-ß-CD showed three strong intermolecular hydrogen bonds. Moreover, the guest inclusion process made from a wider CD rim presented lower energies (interaction and Gibbs free energy) in comparison to the inclusion made by a narrower CD rim in both gas and aqueous phases. This difference in energies of drug/CM-ß-CD inclusion complexes is probably a measure of chiral discrimination, which results in the separation of the enantiomers and the distinct separation factors as observed in previous experimental findings. Comparing the experimental results of the separation of 4-OH-Prop enantiomers by capillary electrophoresis (CE), the proposed theoretical model demonstrated good capability to predict chiral separation of 4-OH-Prop enantiomers as well as the qualitative estimative of chiral recognition mechanism.