Gabriella Massolini

Isolation and characterization of bioactive compounds from plant resources: The role of analysis in the ethnopharmacological approach

The phytochemical research based on ethnopharmacology is considered an effective approach in the discovery of novel chemicals entities with potential as drug leads. Plants/plant extracts/decoctions, used by folklore traditions for treating several diseases, represent a source of chemical entities but no information are available on their nature. Starting from this viewpoint, the aim of this review is to address natural-products chemists to the choice of the best methodologies, which include the combination of extraction/sample preparation tools and analytical techniques, for isolating and characterizing bioactive secondary metabolites from plants, as potential lead compounds in the drug discovery process. The work is distributed according to the different steps involved in the ethnopharmacological approach (extraction, sample preparation, biological screening, etc.), discussing the analytical techniques employed for the isolation and identification of compound/s responsible for the biological activity claimed in the traditional use (separation, spectroscopic, hyphenated techniques, etc.). Particular emphasis will be on herbal medicines applications and developments achieved from 2010 up to date.

Separation of purine and pyrimidine bases and nucleosides by hydrophilic interaction chromatography

The separation of 12 model compounds chosen among purine and pyrimidine bases and nucleosides was studied by using hydrophilic interaction chromatography (HILIC). The compounds investigated were small molecules with relevant properties for biomedical and pharmaceutical studies. The mixture of pyrimidines and purines was applied on a ZIC-HILIC 150 x 2.1 mm, 5 microm, and two TSKgel Amide-80 150 x 2.0 mm, 5 microm and 3 microm particle size columns. The retention of the analytes was studied by varying ACN%, ammonium formate concentration, pH, and column temperature. The results obtained confirmed the elution order of nucleobases, nucleosides, and nucleotides based on their hydrophobicity. The retention mechanism of the columns was studied considering the models used for describing partitioning and surface adsorption. The influence on retention of chromatographic conditions (ACN%, salt concentration, pH, and temperature) was described and discussed for both columns. The optimization of the conditions studied allowed to assess a gradient method for the separation of the 12 analytes. The developed method is a valuable alternative to existing methods for the separation of the compounds concerned.

Validation of a RP-LC method for the simultaneous determination of isoniazid, pyrazinamide and rifampicin in a pharmaceutical formulation.

A simple and accurate liquid chromatographic method was developed and validated for estimation of isoniazid (ISN), pyrazinamide (PYR) and rifampicin (RIF) in combined dosage forms. Drugs were chromatographed on a reverse phase C18 column using a mobile phase gradient and monitored at the corresponding maximum of each compounds. Peaks were identified with retention time as compared with standards and confirmed with characteristic spectra using diode-array detector. Solution concentrations were measured on a weight basis to avoid the use of an internal standard. The method does not require any specific sample preparation except the use of a guard column. The method is linear (r(2)>0.999), precise (RSD%: 0.50% for ISN, 0.12% for PYR and 0.98% for RIF), accurate (overall average recovery yields: 98.55% for ISN, 98.51 for PYR and 98.56% for RIF) and selective. Due to its simplicity and accuracy the method is suitable for routine quality control analysis of antitubercolosis combination dosage form.

Surface initiated molecularly imprinted polymer films: a new approach in chiral capillary electrochromatography

A new generation of imprinted composite particles was tested as capillary electrochromatography stationary phase. Silica particles characterised by a well defined particle size (10 µm diameter), shape and pore system (1000 A) were modified with an azoinitiator and subsequently used to graft molecularly imprinted polymers targeted to bind L-phenylalanine anilide. Fused silica capillaries were packed over a length corresponding to 8 cm, using a pneumatic amplification pump, and the stationary phase thus obtained was tested with respect to its electrochromatographic performance. The electroendosmotic flow mobility was evaluated with respect to both the different content of polymer on the silica particle surface and different operating pH values. The dependence of various parameters, namely the analyte concentration, the polymer layer thickness and the pH of the mobile phase on the enantioselectivity was investigated. These CEC capillaries showed enantioselectivity comparable with that showed in LC, and exhibited improved performance in terms of plate number N1 (∼13 000), selectivity α (∼1.5), analysis time (<3 min), inter–intraday and intercapillary reproducibility. We expect this approach to result in a new generation of robust, tailor-made chiral or affinity stationary phases for CEC.

Capillary electrophoresis investigation of a partially unfolded conformation of β2-microglobulin

Dialysis‐related amyloidosis is a disease in which partial unfolding of β2‐microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of β2‐microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full‐length β2‐microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full‐length β2‐microglobulin.

Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17+] and from the same cell line transfected with the corresponding empty vector [GPR17(-)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)-IAM and GPR17(-)-IAM stationary phases. Frontal chromatography experiments on both GPR17(+)-IAM and GPR17(-)-IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)-IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants (K(d)) of three ligands-the antagonist cangrelor (formerly AR-C69931MX, a P2Y(12)/P2Y(13) antagonist), MRS2179 (a P2Y(1) receptor antagonist), and the agonist UDP-all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated K(d) values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)-IAM in ranking affinity studies for the selection of new potential candidates.

Resveratrol and its metabolites bind to PPARs

Resveratrol, a modulator of several signaling proteins, can exert off‐target effects involving the peroxisome proliferator‐activated receptor (PPAR) transcription factors. However, evidence for the direct interaction between this polyphenol and PPARs is lacking. Here, we addressed the hypothesis that resveratrol and its metabolites control aspects of PPAR transcriptional activity through direct interaction with PPARs. Bioaffinity chromatographic studies with the immobilized ligand‐binding domains (LBDs) of PPARγ and PPARα and isothermal titration calorimetry allowed the binding affinities of resveratrol, resveratrol 3‐O‐glucuronide, resveratrol 4‐O‐glucuronide, and resveratrol 3‐O‐sulfate to both PPAR‐LBDs to be determined. Interaction of resveratrol, resveratrol 3‐O‐glucuronide, and resveratrol 4‐O‐glucuronide with PPARγ‐LBD occurred with binding affinities of 1.4, 1.1, and 0.8 μM, respectively, although only resveratrol bound to the PPARα‐LBD with a binding affinity of 2.7 μM. Subsequently, X‐ray crystallographic studies were carried out to characterize resveratrol binding to the PPARγ‐LBD at the molecular level. The electron density map from the crystal structure of the complex between PPARγ‐LBD and resveratrol revealed the presence of one molecule of resveratrol bound to the LBD of PPARγ, with the ligand occupying a position close to that of other known PPARγ ligands. Transactivation assays were also performed in HepG2 cells, with the results showing that resveratrol was not a PPAR agonist but instead was able to displace rosiglitazone from PPARγ and Wy‐14643 from PPARα with IC50 values of (27.4±1.8) μM and (31.7±2.5) μM, respectively. We propose that resveratrol acts as a PPAR antagonist through its direct interaction with PPARγ and PPARα.

Immobilized trypsin on epoxy organic monoliths with modulated hydrophilicity: Novel bioreactors useful for protein analysis by liquid chromatography coupled to tandem mass spectrometry

The development of epoxy organic monoliths with modulated hydrophilicity for the preparation of novel trypsin-based microreactors is reported. Porous polymer monoliths have been prepared using methacrylate chemistry triggered by γ-ray irradiation. In situ polymerization has been optimized and extended to medium and high polymer densities using glycidyl methacrylate (GMA) as reactive monomer as well as to the hydrophilic nature of the co-monomers (glyceryl monomethacrylate, GlyMA and acrylamide, AMD). Enzyme immobilization was smoothly achieved by passing a buffered trypsin solution through the columns kept at room temperature. The activities of the immobilized enzyme were characterized by the apparent Michaelis constant (K(m)) and the apparent maximum velocity (V(max)) of the reaction using a non chromogenic, low-molecular mass substrate N-α-benzoyl-l-arginine ethyl ester (BAEE). For the kinetic constants determination a new off-line chromatographic procedure was developed on purpose. The most efficient IMERs were obtained by immobilizing trypsin on monolithic skeleton prepared with hydrophilic monomers (GlyMA and AMD). One of the most promising bioreactor was applied to the digestion of model proteins with different molecular weight and complexity such as human serum albumin (HSA), β-casein and ribonuclease B (RNase B), and the produced peptides were analyzed by liquid chromatography-mass spectrometry. Using a digestion time of only 25 min the proteins were recognized by the database with satisfactory sequence coverage, which was 78.22, 49.76 and 80.68% for HSA, β-casein and RNase B, respectively.

Egg yolk riboflavin binding protein as a new chiral stationary phase in high-performance liquid chromatography

A chiral stationary phase for high-performance liquid chromatography based on hen egg yolk riboflavin binding protein is introduced. The purified protein was immobilized on activated 5NH2 Nucleosil silica. Chiral acidic, basic and uncharged drugs were chromatographed and the influence of the mobile phase parameters on the retention times and enantioselectivity was studied. Thirteen out of the twenty compounds tested were partially or baseline resolved. These encouraging preliminary results suggest that this chiral stationary phase may be applicable to a wide range of drug enantiomers in the reversed-phase mode.

Target-Based Drug Discovery: the Emerging Success of Frontal Affinity Chromatography Coupled to Mass Spectrometry

Frontal affinity chromatography coupled to mass spectrometry (FAC–MS) has been reported as a potential method for screening compound mixtures against immobilized target proteins. The potentiality of this analytical approach is described and illustrated with a number of examples based on targets of pharmaceutical interest.