Charalambos Fotakis

Development and validation of a chemiluminogenic method for the evaluation of antioxidant activity of hydrophilic and hydrophobic antioxidants

A sensitive and simple procedure is described for evaluating the antioxidant activity of 21 known hydrophilic and hydrophobic compounds by using the chemiluminogenic reaction of lucigenin with hydrogen peroxide. The method was validated for linearity, limit of detection, precision, additivity and ruggedness and compared to the DPPH method. Furthermore, the IC(50) values of all compounds were calculated and compared by both methods and results are discussed on the basis of structure-antioxidant activity relationship. Among hydrophilic antioxidants, phenolic compounds with adjacent hydroxyl groups (gallic acid, caffeic acid, pyrocatechol, (+/-)-catechin, oleuropein) were found to be efficient antioxidants in chemiluminescence method with IC(50) values at the sub-micromolar range, while phenolic compounds without adjacent hydroxyl groups (p-coumaric acid, BHT, BHA, resorcinol) exhibited weak activity with IC(50) values>10microM. Alpha-tocopherol was the most efficient hydrophobic antioxidant (IC(50)=6.7microM) while oleic and linoleic acids were found to be very poor antioxidants (IC(50)>300microM). Finally the method was applied to the estimation of antioxidant activity of complex samples such as olive oils. To the best of our knowledge, this is the first time that total antioxidant activity can be determined directly in olive oil and not in aqueous extracts.

Flow-Based Methods with Chemiluminescence Detection for Food and Environmental Analysis: A Review

This paper presents an overview of flow-based methods in food and environmental analysis using chemiluminescence (CL) detection covering the period from 2005 to the present. The review discussses both automated flow methods of analysis [such as flow-injection analysis (FIA), sequential-injection analysis (SIA) and their variants] and separation techniques [liquid chromatography (LC) and capillary electrophoresis (CE) coupled to CL detection]. The most widely used CL reactions are presented together with representative applications in food and environmental analysis (determination of naturally occurring compounds, contaminants, additives as well as determination of inorganic and organic compounds).

Comparative Biophysical Studies of Sartan Class Drug Molecules Losartan and Candesartan (CV-11974) with Membrane Bilayers

The interactions of the antihypertensive AT(1) antagonists candesartan and losartan with membrane bilayers were studied through the application of DSC, Raman, and solid state (31)P NMR spectroscopies. (1)H and (13)C NMR resonances of candesartan were assigned on the basis of 1D and 2D NMR spectroscopy. A (31)P CP NMR broadline fitting methodology in combination with ab initio computations was implemented and, in conjunction with DSC and Raman results, provided valuable information regarding the perturbation, localization, orientation, and dynamic properties of the drugs in membrane models. In particular, results indicate that losartan anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup, whereas candesartan has less definite localization spanning from water interface toward the mesophase and upper segment of the hydrophobic region. Both sartan molecules decrease the mobilization of the phospholipids alkyl chains. Losartan exerts stronger interactions compared with candesartan, as depicted by the more prominent thermal, structural, and dipolar (1)H-(31)P changes that are caused in the lipid bilayers. At higher concentrations, candesartan strengthens the polar interactions and induces increased order at the bilayer surface. At the highest concentration used (20 mol %), only losartan induces formation of microdomains attributed to the flexibility of its alkyl chain. These results in correlation to reported data with other AT(1) antagonists strengthen the hypothesis that this class of molecules may approach the active site of the receptor by insertion in the lipid core, followed by lateral diffusion toward the binding site. Further, the similarities and differences of these drugs in their interactions with lipid bilayers establish, at least in part, their pharmacological properties.

Interactions at the bilayer interface and receptor site induced by the novel synthetic pyrrolidinone analog MMK3

This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 with the model membrane system of dipalmitoylphosphatidylcholine (DPPC) and the receptor active site. MMK3 has been designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT(1)). Its low energy conformers were characterized by 2D rotating-frame Overhauser effect spectroscopy (ROESY) in combination with molecular dynamics (MD) simulations. Docking study of MMK3 shows that it fits to the AT(1) receptor as SARTANs, however, its biological activity appears to be lower. Thus, differential scanning calorimetry (DSC), Raman spectroscopy and small angle X-ray scattering (SAXS) experiments on the interaction of MMK3 with DPPC bilayers were carried out and results demonstrate that the drug is well incorporated into the membrane leaflets and furthermore causes partial bilayer interdigitation, although less effective than SARTANs. Thus, it appears that the nature of the bilayer matrix and the stereoelectronic active site requirements of the receptor are responsible for the low bioactivity of MMK3.

Luminescent Methods in the Analysis of Untreated Edible Oils: A Review

The present review describes the application of luminescent methods in the analysis of edible oils without any pretreatment such as extraction prior to analysis. Emphasis has been given to applications of chemiluminescence and fluorescence assays for determining quality parameters of edible oils, such as oxidative stability, antioxidant activity, and lipid hydroperoxides content, as well as classification or adulteration of vegetable oils.

Modified DPPH and ABTS Assays to Assess the Antioxidant Profile of Untreated Oils

This research dwells on two widely used spectrophotometric methods, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, which assess the free radical scavenging activity (RSA) of natural samples and standard compounds. In particular, these assays were modified in order to simplify the evaluation of RSA of untreated edible oils, as well as to assess the antioxidant profile of oils’ hydrophilic and lipophilic extracts with the same analytical procedure. A thorough study highlighted the effect of solvents on the DPPH and ABTS methods and resulted in selecting 2-propanol and an ethanol/1-butanol solvent mixture as the reaction solvent for the DPPH method and the ABTS method, respectively. The developed methods were used to evaluate the RSA of 12 antioxidant compounds and 8 edible oils. Then, the contribution of lipophilic and hydrophilic extracts to the total RSA of oils was estimated. The obtained results demonstrate the applicability of the method to routine edible oil analysis.

NMR metabolite profiling of Greek grape marc spirits.

This (1)H NMR based study profiles metabolites in Greek grape marc distillates, tsipouro and tsikoudia. Eightysix samples of indigenous and international varieties, stemming from major vine growing regions of Greece were investigated. The monitoring protocol addressed the global metabolic profile of untreated samples and accomplished the unambiguous assignment of 35 metabolites. NMR spectra were acquired by applying the robust, sensitive and rapid WET1D NMR pulse sequence, which succeeded to unveil the presence of minor compounds in a high ethanol matrix. PCA classified the samples according to their provenance, incorporating also information related to the variety, vintage year and production process within each formed regional assembly. Metabolites such as fusel alcohols, polyols, ethyl esters, mono- and di-saccharides were associated with the classification of samples. OPLS-DA ascribed to samples of common regional entity characteristic genotypic metabolites and probed to the potential influence of the vintage effect. Finally, metabolite profiling underlined the influence of the fermentation and distillation procedures.

NMR metabolic fingerprinting and chemometrics driven authentication of Greek grape marc spirits.

NMR metabolomics was used to investigate 57 Greek spirits of four indigenous and four international varieties from Macedonia (tsipouro) and Crete (tsikoudia) to establish their authenticity. The metabolic profile of Romeiko, Malvasia, Xinomavro, Sangiovese and Nebbiolo varieties was assessed for the first time. The WET1D sequence was used to improve sensitivity and unveil minor metabolites. PCA was applied to delineate the provenance of samples and associate metabolites with distinct varietal characteristics, such as the acidity of Sangiovese, the overripe grapes harvesting of Romeiko, the intense body of Cabernet Sauvignon, the light body of Xinomavro and the glutamic acid for Malvasia. The migration of Cabernet Sauvignon from north Greece to Crete was framed. Monitoring multi-varietal spirits introduced 2-vinylethanol as a marker for yeast selection. OPLS-DA was applied to samples from the same vineyard, thus highlighting genotypic markers. Consequently, the findings address the concepts of typicity and traceability in grape marc spirits.

Comparative study of the AT1 receptor prodrug antagonist candesartan cilexetil with other sartans on the interactions with membrane bilayers

Drug-membrane interactions of the candesartan cilexetil (TCV-116) have been studied on molecular basis by applying various complementary biophysical techniques namely differential scanning calorimetry (DSC), Raman spectroscopy, small and wide angle X-ray scattering (SAXS and WAXS), solution ¹H and ¹³C nuclear magnetic resonance (NMR) and solid state ¹³C and ³¹P (NMR) spectroscopies. In addition, ³¹P cross polarization (CP) NMR broadline fitting methodology in combination with ab initio computations has been applied. Finally molecular dynamics (MD) was applied to find the low energy conformation and position of candesartan cilexetil in the bilayers. Thus, the experimental results complemented with in silico MD results provided information on the localization, orientation, and dynamic properties of TCV-116 in the lipidic environment. The effects of this prodrug have been compared with other AT₁ receptor antagonists hitherto studied. The prodrug TCV-116 as other sartans has been found to be accommodated in the polar/apolar interface of the bilayer. In particular, it anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup spanning from water interface toward the mesophase and upper segment of the hydrophobic region. In spite of their localization identity, their thermal and dynamic effects are distinct pointing out that each sartan has its own fingerprint of action in the membrane bilayer, which is determined by the parameters derived from the above mentioned biophysical techniques.

Development of a CP 31P NMR Broadline Simulation Methodology for Studying the Interactions of Antihypertensive AT1 Antagonist Losartan with Phospholipid Bilayers

A cross-polarization (CP) (31)P NMR broadline simulation methodology was developed for studying the effects of drugs in phospholipids bilayers. Based on seven-parameter fittings, this methodology provided information concerning the conformational changes and dynamics effects of losartan in the polar region of the dipalmitoylphosphatidylcholine bilayers. The test molecule for this study was losartan, an antihypertensive drug known to exert its effect on AT(1) transmembrane receptors. The results were complemented and compared with those of differential scanning calorimetry, solid-state (13)C NMR spectroscopy, Raman spectroscopy, and electron spin resonance. More specifically, these physical chemical methodologies indicated that the amphipathic losartan molecule interacts with the hydrophilic-head zone of the lipid bilayers. The CP (31)P NMR broadline simulations showed that the lipid molecules in the bilayers containing losartan displayed greater collective tilt compared to the tilt displayed by the load-free bilayers, indicating improved packing. The Raman results displayed a decrease in the trans/gauche ratio and increased intermolecular interactions of the acyl chains in the liquid crystalline phase. Additional evidence, suggesting that losartan possibly anchors in the realm of the headgroup, was derived from upfield shift of the average chemical shift sigma(iso) of the (31)P signal in the presence of losartan and from shift of the observed peak at 715 cm(-1) attributed to C-N stretching in the Raman spectra.