Malvina G. Orkoula

Investigation of the neuroprotective action of saffron (Crocus sativus L.) in aluminum-exposed adult mice through behavioral and neurobiochemical assessment.

In the present study, the possible reversal effects of saffron against established aluminum (Al)-toxicity in adult mice, were investigated. Control, Al-treated (50 mg AlCl(3)/kg/day diluted in the drinking water for 5 weeks) and Al+saffron (Al-treatment as previously plus 60 mg saffron extract/kg/day intraperitoneally for the last 6 days), groups of male Balb-c mice were used. We assessed learning/memory, the activity of acetylcholinesterase [AChE, salt-(SS)/detergent-soluble(DS) isoforms], butyrylcholinesterase (BuChE, SS/DS isoforms), monoamine oxidase (MAO-A, MAO-B), the levels of lipid peroxidation (MDA) and reduced glutathione (GSH), in whole brain and cerebellum. Brain Al was determined by atomic absorption spectrometry, while, for the first time, crocetin, the main active metabolite of saffron, was determined in brain after intraperitoneal saffron administration by HPLC. Al intake caused memory impairment, significant decrease of AChE and BuChE activity, activation of brain MAO isoforms but inhibition of cerebellar MAO-B, significant elevation of brain MDA and significant reduction of GSH content. Although saffron extract co-administration had no effect on cognitive performance of mice, it reversed significantly the Al-induced changes in MAO activity and the levels of MDA and GSH. AChE activity was further significantly decreased in cerebral tissues of Al+saffron group. The biochemical changes support the neuroprotective potential of saffron under toxicity.

Quantitative Analysis of Sulfated Calcium Carbonates Using Raman Spectroscopy and X-ray Powder Diffraction

A non-destructive method based on the use of Raman spectroscopy (RS) for the determination of the percentage of gypsum in sulfated marble is presented. The Raman spectra of well mixed powder samples of calcite–aragonite, calcite–gypsum and gypsum–aragonite pairs of mixtures were recorded and the characteristic bands at 280 cm - 1 for calcite, 205 cm - 1 for aragonite and 412 cm - 1 for gypsum were used as the basis for the quantitative analysis of specimens in which the most stable calcium carbonate phases, calcite and aragonite, were present. The detection limits were found to be 0.3 mol% for calcite, 0.5 mol% for aragonite and 0.6 mol% for gypsum. For samples containing only one calcium carbonate phase the use of the strong and sharp Raman band at 1085 cm - 1 , common for aragonite and calcite, together with the intensity of the Raman peak at 1006 cm - 1 for gypsum, yielded lower detection limits: calcite 0.1, aragonite 0.1 and gypsum 0.05 mol%. The analysis by RS was compared with X-ray powder diffraction (XRD). In this analysis, the calibration curves were constructed using the relative intensities corresponding to the 113, the 111 and the 12 reflections of the calcite, aragonite and gypsum, respectively. The detection limits for calcite, aragonite and gypsum were 4, 5 and 1–2 mol%, respectively. The potential of using RS for a point-by-point analysis (‘mapping’) of a surface by focusing the laser beam on the selected spots was also demonstrated on a marble sample removed from Athens National Garden, exposed in the open air.

Analysis and stability of polymorphs in tablets: The case of Risperidone.

Identification of the crystal phase of an active pharmaceutical ingredient (API) in a pharmaceutical tablet is of outmost importance since different polymorphs exhibit different physicochemical properties. Furthermore, some of the crystal phases are protected by patents. Identification of Risperidone polymorph A in film coated commercial tablets was attempted using IR spectroscopy, Raman spectroscopy and X-ray powder diffraction (XRPD). The stability of this polymorph through time and during the manufacturing process was also examined. The inability of IR and Raman techniques to identify the presence of polymorph A in the tablets, despite their lower detection limits for Risperidone, left the XRPD as the only technique that could be used for identifying the presence of Risperidone A against the other crystal phases in the presence of the excipients. Polymorph A was proved to be stable during the manufacturing process and after a storage period of 2 years.

Quantitative non-destructive determination of salicylic acid acetate in aspirin tablets by Raman spectroscopy

Laser Raman spectroscopy was used for the quantitative determination of aspirin in aspirin-maize starch tablets. A calibration curve was constructed from spectra obtained from tablets with known quantities of aspirin and starch. The calibration curve is given from the relationship: I(552)/I(478) = (W(aspirin)/W(starch)) x 4.21, where I(552) and I(478) are the relative Raman intensities for the 552 and 478 cm(-1) Raman shift, respectively. W(aspirin) and W(starch) represent the weight of aspirin and starch in a pellet.

Non-destructive quantitative analysis of risperidone in film-coated tablets

A simple, non-destructive, methodology based on FT-Raman spectroscopy was developed for the quantitative analysis of risperidone in commercially available film-coated tablets. A simple linear regression model was constructed based on standard tablets, prepared using the same manufacturing process as the commercially available. The tablets contained 0.27, 0.54, 1.08, 1.62, 2.16, 3.24 and 4.32 wt% risperidone. The most prominent Raman vibration of the active pharmaceutical ingredient at 1533 cm(-1), recorded using a home-made rotating system, was plotted against concentration. The model was tested on commercial film-coated tablets. The results were compared against those obtained by application of HPLC on the same samples.

A quantitative bioapatite/collagen calibration method using Raman spectroscopy of bone

Numerous calibration models were developed and tested for the quantitative analysis of collagen and bioapatite in bone using Raman spectroscopy. The ν1 phosphate vibration at 960 cm(-1) was used as indicator of the content of bioapatite while for collagen three markers were used: the C-H2 band at 2940 cm(-1) , the amide I band at 1667 cm(-1) and the vibrations of proline and hydroxyproline at 855 and 878 cm(-1) , respectively. Also a calibration model based on the PLS algorithm was developed, too. Validation of the derived calibration models indicated that the model that makes use of the height ratio of the peaks 960/(855+878) exhibits the best accuracy.

Wettability of CaCO3 surfaces

Abstract The wettability of calcium carbonate, an important parameter in the evaluation of the interaction characteristics of materials used in conservation, was examined by measurements with the Wilhelmy plate technique. Heterogeneous surfaces consisting of glass and calcium carbonate were examined. Stable calcium carbonate layers were deposited on microscope glass slides using supersaturated solutions in which the equimolar concentrations of total calcium and total carbonate used ranged between 0.1 and 0.4 M, over a pH range between 9 and 12 and temperature between 25 and 80°C. In all cases amorphous calcium carbonate was deposited, the extent of surface coverage increasing with increasing supersaturation and temperature. Higher surface coverage of the glass slides with calcium carbonate yielded lower contact angles suggesting increased wettability of the heterogeneous surface.

Alarm Photosynthesis: Calcium Oxalate Crystals as an Internal CO2 Source in Plants

A new photosynthetic path named “alarm photosynthesis” uses mesophyll calcium oxalate crystals as the CO2 source when stomata are closed, providing adaptive advantages under drought conditions. Calcium oxalate crystals are widespread among animals and plants. In land plants, crystals often reach high amounts, up to 80% of dry biomass. They are formed within specific cells, and their accumulation constitutes a normal activity rather than a pathological symptom, as occurs in animals. Despite their ubiquity, our knowledge on the formation and the possible role(s) of these crystals remains limited. We show that the mesophyll crystals of pigweed (Amaranthus hybridus) exhibit diurnal volume changes with a gradual decrease during daytime and a total recovery during the night. Moreover, stable carbon isotope composition indicated that crystals are of nonatmospheric origin. Stomatal closure (under drought conditions or exogenous application of abscisic acid) was accompanied by crystal decomposition and by increased activity of oxalate oxidase that converts oxalate into CO2. Similar results were also observed under drought stress in Dianthus chinensis, Pelargonium peltatum, and Portulacaria afra. Moreover, in A. hybridus, despite closed stomata, the leaf metabolic profiles combined with chlorophyll fluorescence measurements indicated active photosynthetic metabolism. In combination, calcium oxalate crystals in leaves can act as a biochemical reservoir that collects nonatmospheric carbon, mainly during the night. During the day, crystal degradation provides subsidiary carbon for photosynthetic assimilation, especially under drought conditions. This new photosynthetic path, with the suggested name “alarm photosynthesis,” seems to provide a number of adaptive advantages, such as water economy, limitation of carbon losses to the atmosphere, and a lower risk of photoinhibition, roles that justify its vast presence in plants.

Effect of hydrazine based deproteination protocol on bone mineral crystal structure.

In several bone deproteination protocols the chemical agent used for protein cleavage is hydrazine. The effect of hydrazine deproteination method on the crystal size and crystallinity of the bone mineral was studied. Bovine bones were subjected to this protocol and the crystal size and crystallinity of the remaining bone mineral were determined by X-ray Diffraction (XRD), by measuring the width at the half of the maximum intensity of the (002) reflection. It was found that hydrazine deproteination induces noteworthy increase of crystal size and crystallinity. The effect was enhanced by increasing hydrazine temperature from 25 to 55°C. Furthermore, infrared spectroscopy revealed that hydrazine facilitates the removal of carbonate and acid phosphate ions from bone mineral. It is proposed that the mechanism of modification of crystal size and crystallinity lies on the removal of these ions thus, resulting in crystal re-organization.

Validation of a direct non-destructive quantitative analysis of amiodarone hydrochloride in Angoron® formulations using FT-Raman spectroscopy

Raman spectroscopy was applied for the direct non-destructive analysis of amiodarone hydrochloride (ADH), the active ingredient of the liquid formulation Angoron((R)). The FT-Raman spectra were obtained through the un-broken as-received ampoules of Angoron((R)). Using the most intense vibration of the active pharmaceutical ingredient (API) at 1568cm(-1), a calibration model, based on solutions with known concentrations, was developed. The model was applied to the Raman spectra recorded from three as-purchased commercial formulations of Angoron((R)) having nominal strength of 50mgml(-1) ADH. The average value of the API in these samples was found to be 48.56+/-0.64mgml(-1) while the detection limit of the proposed technique was found to be 2.11mgml(-1). The results were compared to those obtained from the application of HPLC using the methodology described in the European Pharmacopoeia and found to be in excellent agreement. The proposed analytical methodology was also validated by evaluating the linearity of the calibration line as well as its accuracy and precision. The main advantage of Raman spectroscopy over HPLC method during routine analysis is that it is considerably faster and no solvent consuming. Furthermore, Raman spectroscopy is non-destructive for the sample. However, the detection limit for Raman spectroscopy is much higher than the corresponding for the HPLC methodology.