Giampiero Pagliuca

Potent Inhibition of Human Phosphodiesterase-5 by Icariin Derivatives

Plant extracts traditionally used for male impotence (Tribulus terrestris, Ferula hermonis, Epimedium brevicornum, Cinnamomum cassia), and the individual compounds cinnamaldehyde, ferutinin, and icariin, were screened against phosphodiesterase-5A1 (PDE5A1) activity. Human recombinant PDE5A1 was used as the enzyme source. Only E. brevicornum extract (80% inhibition at 50 microg/mL) and its active principle icariin (1) (IC50 5.9 microM) were active. To improve its inhibitory activity, 1 was subjected to various structural modifications. Thus, 3,7-bis(2-hydroxyethyl)icaritin (5), where both sugars in 1 were replaced with hydroxyethyl residues, potently inhibited PDE5A1 with an IC50 very close to that of sildenafil (IC50 75 vs 74 nM). Thus, 5 was 80 times more potent than 1, and its selectivity versus phosphodiesterase-6 (PDE6) and cyclic adenosine monophosphate-phosphodiesterase (cAMP-PDE) was much higher in comparison with sildenafil. The improved pharmacodynamic profile and lack of cytotoxicity on human fibroblasts make compound 5 a promising candidate for further development.

INVESTIGATION OF 4‐METHYL STEROLS FROM CULTURED DINOFLAGELLATE ALGAL STRAINS

The marine dinoflagellates Prorocentrum micans, Gonyaulax polyedra, Gymnodinium sp., and Alexandrium tamarense, collected from the Adriatic Sea during red‐tide blooms, were cultured to investigate the 4‐methyl sterol constituents. To ascertain a possible influence of cell age on the 4‐methyl sterol content, for one strain (Gymnodinium sp.)we investigated the composition of these constituents at exponential and stationary growing phases. The lipid material extracted with acetone from the lyophilized algal samples was fractionated by thin‐layer chromatography. The 4‐methyl sterols recovered from the layer were converted into the corresponding OTMS derivatives. Nine of 11 constituents were identified by gas chromatography and gas chromatography‐mass spectrometry; only two minor constituents were characterized by their gas chromatographic parameters. All free methyl sterols identified in the algal samples had been detected previously in various dinoflagellates. The 4‐methyl sterol fractions generally contained very few constituents. Except for the Gymnodinium sp. sample, collected at the exponential growing phase (GyD2 exp), which contains 4,24‐dimethylcholestan‐3‐ol as a unique constituent, dinosterol was the major component. Moreover, 4,24‐ethylcholestan‐3‐ol was also an important constituent of both Prorocentrum and Gonyaulax strains, whereas considerable amounts of dinostanol characterized all the Gymnodinium sp. strains. In addition, the latter contained several minor constituents such as 4‐methylcholestan‐3‐ol, 4,24‐dimethylcholesta‐22‐en‐3‐ol, and 4‐methyl‐24‐ethylcholestan‐3‐ol. 4‐Methyl‐24‐methylene‐cholestan‐3‐ol was a constituent of the Gymnodinium sp. sample, collected at the stationary growing phase (GyD2 stat)only, whereas 4‐methylgorgostanol was identified only in the Alexandrium tamarense Gt4 strain. Except for 4‐methyl‐24‐ethylcholesta‐8(14)‐en‐3‐ol, all the methyl sterol constituents from our algae show a saturated polynuclear system. The pathways by which side‐chain modifications occur in dinoflagellate 4‐methyl sterols are considered, and a map of the fragmentation pattern of the trimethylsilyl‐4‐methyl sterols under electronic impact is also reported.

Organophosphorus pesticides residues in Italian raw milk

Organophosphorus pesticides (OPPs), widely used in agriculture, can cause toxic effects to humans and animals. The main purpose of the present work was to determine the contamination in raw milk by the main organophosphorus pesticides used in Italy and to evaluate the opportunity to start specific procedures of risk management along the milk production chain. The samples, collected in 4 Italian dairy plants directly from the tank trucks during the delivering, were representative of 920 tonnes of raw milk. The isolation of the OPPs (acephate, chlorpyriphos, chlorpyriphos-methyl, diazinon, methamidophos, methidathion, phorate, pirimiphos-methyl) was performed by liquid partition followed by clean-up with solid phase extraction. The analyses were carried out by dual column gas chromatography using two nitrogen-phosphorus detectors. Among the 135 samples analysed, 37 were positive in traces and 10 showed an OPP contamination ranging from 5 to 18 microg/kg. The higher results were recorded in the samples collected during the autumn-winter period. The main pollutants detected were acephate and chlorpyriphos. In every positive sample found, the OPP contamination was lower than the maximum residue level (MRL) fixed by the European Commission.

Perfluoroalkyl substances in human milk: a first survey in Italy.

Due to their widespread diffusion, perfluoroalkyl substances (PFASs) have been frequently found in the environment and in several animal species. It has been demonstrated that they can easily reach also humans, mainly through diet. Being lactation a major route of elimination of these contaminants, their occurrence in human milk is of particular interest, especially considering that it generally represents the unique food source for newborns. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), the two most important compounds of this family, have been frequently found in human milk at variable concentrations, but still limited data are available. The present study, the first conducted in Italy capable to detect these pollutants at ultra-trace levels by UPLC-MS/MS, confirmed the role of lactation as a relevant source of exposure for breastfed children. The measured concentrations ranged between 15 and 288 ng/L for PFOS and between 24 and 241 ng/L for PFOA. Moreover, mean concentrations and frequencies of both analytes resulted higher in milk samples provided by primiparous women, suggesting that the risk of intake might be higher for first-borns. Finally, comparing these results with previous data, PFOS gradual decrease over time since year 2000 was confirmed.

Brain distribution of ribavirin after intranasal administration

Ribavirin has proved to be effective in vitro against several RNA viruses responsible for encephalitis in humans and animals. However, the in vivo efficacy towards the cerebral viral load seems to be limited by the blood-brain barrier. Since the nose-to-brain pathway has been indicated for delivering drugs to the brain, we investigated here the distribution of ribavirin in the central nervous system (CNS) after intranasal administration. We first tested in vitro ribavirin diffusion from an aqueous solution across a biological membrane, using Franz cells and rabbit nasal mucosa. About 35% of ribavirin permeated in 4 h across the mucosa, after reaching steady-state flux in less than 30 min. In the first in vivo experiment, ribavirin aqueous solution was administered intranasally to Sprague Dawley rats (10 mg/kg). Animals were sacrificed at 10, 20 or 30 min after administration to collect brain areas (cerebellum, olfactory bulb, cerebral cortex, basal ganglia and hippocampus) and biological fluids (cerebrospinal fluid and plasma). Ribavirin, quantified by LC-MS/MS spectrometry, was detected at each time point in all compartments with the highest concentration in olfactory bulb and decreasing in rostro-caudal direction. Two subsequent in vivo experiments compared the nasal route (ribavirin solution) with the intravenous one and the nasal administration of ribavirin solution with ribavirin powder (10 mg/kg). It was found that 20 min after administration, ribavirin concentration in olfactory bulb was similar after intravenous or nasal administration of the ribavirin solution, whereas the powder led to significantly higher levels. Ribavirin was also present in deeper compartments, such as basal ganglia and hippocampus. Even if the mechanisms involved in ribavirin nose-to-brain transport are not clear, these results suggest a rapid extracellular diffusive flux from the nasal epithelium to the olfactory bulb and different CNS areas.

Development of an analytical strategy based on liquid chromatography–high resolution mass spectrometry for measuring perfluorinated compounds in human breast milk: Application to the generation of preliminary data regarding perinatal exposure in France

Perfluorinated compounds (PFCs) are man-made chemicals for which endocrine disrupting properties and related possible side effects on human health have been reported, particularly in the case of an exposure during the early stages of development, (notably the perinatal period). Existing analytical methods dedicated to PFCs monitoring in food and/or human fluids are currently based on liquid chromatography coupled to tandem mass spectrometry, and were recently demonstrated to present some limitations in terms of sensitivity and/or specificity. An alternative strategy dedicated to the analysis of fourteen PFCs in human breast milk was proposed, based on an effective sample preparation followed by a liquid chromatography coupled to high resolution mass spectrometry measurement (LC-HRMS). This methodology confirmed the high interest for HRMS after negative ionization for such halogenated substances, and finally permitted to reach detection limits around the pg mL(-1) range with an outstanding signal specificity compared to LC-MS/MS. The proposed method was applied to a first set of 30 breast milk samples from French women. The main PFCs detected in all these samples were PFOS and PFOA with respective median values of 74 (range from 24 to 171) and 57 (range from 18 to 102) pg mL(-1), respectively. These exposure data appeared in the same range as other reported values for European countries.

Investigation of the seasonal variations of fatty acid constituents in selected tissues of the bivalve mollusc Scapharca inaequivalvis (Bruguiere)

Abstract 1. 1. The seasonal variations in the fatty acid composition of the total lipids were studied in four selected tissues of Scapharca inaequivalvis : gills, I; mantle, II; foot together with digestive diverticula, III and haemolymph, IV. 2. 2. Forty-eight main fatty acids were found. Of these 30 were identified through the mass spectra of the corresponding methyl esters and/or by the increase in the gas chromatographic peak with suitable standards. 3. 3. The remaining fatty acids were characterized exclusively by means of the corresponding ECL values. 4. 4. The constituents identified are those that have normally been found in bivalve molluscs and phytoplankton. 5. 5. The qualitative composition of the fatty acid mixtures from I, II and III is very similar and clearly differs from that of IV. 6. 6. Fluctuations in the percentages of the single constituents were observed over the course of the year. 7. 7. A percentage increase in the unsaturated constituents was noted in the cold months. 8. 8. The percentage composition variations in the fatty acids from the lipid extracts of the different tissues and from the whole organism would seem to be linked to the feeding—growth, nonfeeding—maturation cycle of the mollusc.

Lipid Peroxidation in Cancer Cells: Chemical and Physical Studies

Our studies on the biochemical composition and the structural organization of smooth and rough endoplasmic reticulum isolated from Morris hepatomas 9618A and 3924A confirm the results obtained employing the total microsomal fraction. We have definitely established the following facts: (1) Tumor subcellular organelles exhibit the very low degree of peroxidizability that has been shown to be related to the growth rate of the tumor. (2) Associated with such a low susceptibility to peroxidation are (a) changed lipid composition of cellular membranes, whose content in polyunsaturated fatty acid is markedly decreased, and (b) changed static and dynamic properties of the membrane. Previously it was also found that cellular oxy-radical scavenging enzymes are markedly reduced. From these data, it is possible to infer that tumor membranes are altered structurally and functionally in part as the result of an oxy-radical-induced damage that occurs in vivo under conditions of oxygen toxicity. This seems to be supported by recent findings that the spontaneous increase in growth rate of the originally very slow-growing Morris hepatoma 9618A results also in the loss of cytochrome P-450 (an important intramembraneous propagator of lipid peroxidation) as well as of C20:4 and C22:6. Studies performed by GLC and GC-MS on the fatty acid residues of phospholipids of rat liver microsomes show the presence of C20:3-OH and C18:1-OH, but no hydroxyl derivatives of low molecular weight aldehydes. The hydroxyl derivatives of arachidonic acid and linoleic acid are present in much smaller amounts in the microsomes isolated from H9618A and H3924A.

Effect of Essential Oils on the Lipids of the Retina in the Ageing Rat: A Possible Therapeutic Use

Abstract A study has been made on the effects of the dietary administration of a selection of volatile oils from medicinal plants on the polyunsaturated fatty acid composition in the retina of aged (28 month old) rats. Polyunsaturated fatty acids comprised by far the major proportion of the total unsaturated content within the retinal phospholipids with docosahexaenoic acid predominating. The administration daily of 3-9 mg of the essential oils from clove, nutmeg, pepper and thyme over a period of 17 months resulted in the maintenance of very much higher levels of polyunsaturated fatty acids, in particular docosahexaenoic acid, within the retinal phospholipids. This increase occurred mainly at the expense of a reduction in the level of oleic acid. The possible efficacy for the application of the oils from such medicinal plants through their antioxidant capacities in the prevention of age-related macular degeneration is discussed.

Proposal of an analytical method for the study of the oxidation products of membrane lipids

A method of lipid sample preparation for GLC and GC-MS analysis is presented which would seem particularly suitable for studying the chemical composition of the oxidation products of membrane lipids. The method requires small amounts of lipid, is quite rapid and avoids the formation of oxidation artifacts during the different analytical steps. Due to the small quantities of lipid material used it is possible that the method is not rigorously quantitative. Transmethylation of lipids is carried out with methanolic NaBH4 in the presence of NaOH. The reaction is complete in 20 min both on neutral and on polar lipids. In the course of the transmethylation the hydroperoxidic groups are reduced to the corresponding hydroxy groups and can be located through the GC-MS spectra of the corresponding TMS derivatives. The epoxidic rings that may be present are not hydrolyzed. They are located by opening the ring with BF3/MeOH and by GC-MS analysis of the corresponding methoxy-hydroxy derivatives.