Domenico Castaldo

Bioavailability of encapsulated resveratrol into nanoemulsion-based delivery systems.

Different O/W nanoemulsion-based delivery systems were developed in order to optimize the bioavailability of encapsulated resveratrol for potential oral administration. Blank formulations without resveratrol had no negative effect on cell viability or the cytoskeleton structure of Caco-2 cells (XTT viability assay and confocal microscopy). All nanoemulsions were then evaluated based on permeability tests on Caco-2 cells. As a result, the most efficient formulations were lecithin-based nanoemulsions which were able to transport resveratrol through cell monolayers in characteristically shorter times (1-6h) than those required for their metabolization (3-12h), allowing for better preservation of the integrity of the emulsion droplets, thus better protecting the resveratrol molecule. Fluorescence spectroscopy studies confirmed that resveratrol was encapsulated in the inner core of the nanoemulsions, which provides protection against chemical degradation. Furthermore, the developed systems also demonstrated the capability of nanoemulsions in sustained release of resveratrol from dialysis bags compared to the unencapsulated compound.

Pectin methyl esterase from Actinidia chinensis fruits

Abstract Pectin methylesterase (EC 3.1.1.11) from Actinidia chinensis fruit, cv Hayward, consists of two forms (PME1 and PME2) separable by heparin-Sepharose chromatography. The two forms have been purified to homogeneity to respective specific activities of 933 and 974 units mg−1. The most efficient step in the purification procedure was the affinity chromatography on heparin-Sepharose column. The enzymatic preparations exhibit the same Mr in native as well as under denaturing conditions; a value of 57,000 was found in ultracentrifugal experiments. The two forms possess the same isoelectric point of 7.3, but differ in their affinity toward citrus pectin having a Km of 1.82 mg ml−1 for PME1 and 0.76 mg ml−1 for PME2. They also differ in thermostability, where PME1 is more stable than PME2. The two forms are glycoproteins with a different degreeof glycosylation as shown by different retention time on concanavalin A-Sepharose with respect to a glucose gradient.

Determination of estragole, safrole and eugenol methyl ether in food products

Abstract A rapid and effective procedure for the extraction and determination of 4-allylanisole (estragole), 4-allyl-1,3-benzodioxole (safrole) and 4-allyl-1,2-dimetoxybenzene (eugenol methyl ether) in food products was developed. These compounds were isolated from food matrices by employing a simultaneous distillation–extraction (SDE) technique with a Likens and Nickerson apparatus and using dichloromethane as extraction solvent. The method was validated by conducting recovery studies on fortified food products at several concentrations. Recoveries ranged between 94 and 105%. The analyses were performed by capillary gas-chromatography, the compounds identified by retention times and confirmed by GC/MS. The detection limits, determined both in standard solutions and in foods, were 10, 5 and 8 ng/ml for estragole, safrole and eugenol methyl ether, respectively and the calibration curves showed a good linearity for all the three compounds in the concentration range 0.5–25 ppm, with correlation coefficients ranging between 0.996 and 1.000. In a number of successive analyses, the estragole peak area repeatability (RSD) was 0.20 ng/ml, whereas for safrole resulted 0.33 ng/ml and for eugenol methyl ether 0.45 ng/ml. The estragole, safrole and eugenol methyl ether levels were measured in 42 samples, for a total of six product types, including “Pesto” sauce, tomato sauce containing basil, “Cola tasting” beverages, Bologna sausage (polony), Vienna sausage (wurstel) and fresh basil. The levels ranged from below the detection limit up to 19.30 mg/kg for estragole in “Pesto” sauce.

Thermal resistance of pectin methylesterase in tomato juice

Abstract The behaviour of pectin methylesterase (PME, EC.3.1.1.11) was analysed in five new tomato cultivars for juices and tomato products. The thermal inactivation of this enzyme was evaluated in the range 73–88 °C and was found to be exponential. For each cultivar examined the logarithmic values of decimal reduction times plotted against temperature had a classic biphasic pattern featuring a sudden change in slope at temperatures exceeding 78 °C. The z values determined in the range 78–88 °C were much higher than those calculated from the log D T versus T °C in the range 73–88 °C.

Betaines in fruits of Citrus genus plants.

Numerous compounds, many of them osmolytes, were quantified in natural juices and in frozen concentrate juices from fruits of plants of the Citrus genus. L-proline, N-methyl-L-proline (hygric acid), N,N-dimethyl-L-proline (stachydrine), 4-hydroxy-L-prolinebetaine (betonicine), 4-hydroxy-L-proline, γ-aminobutyric acid (Gaba), 3-carboxypropyltrimethylammonium (GabaBet), N-methylnicotinic acid (trigonelline), and choline in the fruit juices of yellow orange, blood orange, lemon, mandarin, bitter orange (Citrus aurantium), chinotto (Citrus myrtifolia), and grapefruit were analyzed by sensitive HPLC-ESI-tandem mass spectrometry procedure. It was found that the most represented osmolytes in the juices, that is, L-proline, stachydrine, and betonicine, can be quantified with minimal sample preparation and short analysis time (about 1 min) also by flow injection analysis (FIA) ESI-MS/MS with the same results as obtained by HPLC ESI-MS/MS. In all of the juices, discrete amounts of choline and trigonelline were present. Conversely, GabaBet was always below detection limits. Notably, N-methyl-L-proline and 4-hydroxy-L-prolinebetaine, which were discovered for the first time in the juice of bergamot (Citrus bergamia Risso et Poit), are also present in all of the citrus juices examined.

An uncommon redox behavior sheds light on the cellular antioxidant properties of ergothioneine.

Ergothioneine (ESH), an aromatic thiol occurring in the human diet and which accumulates in particular cells, is believed to act as an antioxidant. However, its redox mechanism remains unclear and it does not seem to provide any advantage compared to other antioxidants, such as alkylthiols, which are better reducing agents and generally present in cells at higher levels. Here, we investigated by ESI-MS the products of ESH oxidation produced by neutrophils during oxidative burst and, to further elucidate ESH redox behavior, we also analyzed the oxidation products of the reaction of ESH with hypochlorite in cell-free solutions. Indeed, neutrophils are the main source of hypochlorite in humans. Furthermore, we also tested other biologically relevant oxidants, such as peroxynitrite and hydrogen peroxide. Our results indicate that treatment of human neutrophils with phorbol 12-myristate 13-acetate in the presence of ESH leads to a remarkable production of the sulfonated form (ESO3H), a compound never described before, and hercynine (EH), the desulfurated form of ESH. Similar results were obtained when ESH was subjected to cell-free oxidation in the presence of hypochlorite, as well as hydrogen peroxide or peroxynitrite. Furthermore, when the disulfide of ESH was reacted with those oxidants, we found that it was also oxidized, with production of EH and ESO3H, whose amount was dependent on the oxidant strength. These data reveal a unique ESH redox behavior, entirely different from that of alkylthiols, and suggest a mechanism, so far overlooked, through which ESH performs its antioxidant action in cells.

Occurrence of pipecolic acid and pipecolic acid betaine (homostachydrine) in Citrus genus plants.

The presence of pipecolic acid and pipecolic acid betaine, also known as homostachydrine, is herein reported for the first time in Citrus genus plants. Homostachydrine was found in fruits, seeds, and leaves of orange, lemon, and bergamot (Citrus bergamia Risso et Poit). As homostachydrine was not commercially available, as a comparative source, extracts of alfalfa leaves ( Medicago sativa L.) were used, in which homostachydrine is present at high concentration. Then, the results where confirmed by comparison with an authentic standard synthesized and purified starting from pipecolic acid. The synthesized standard was characterized by a ESI-MS/MS study using a 3D ion-trap mass spectrometer. When subjected to MS/MS fragmentation in positive ion mode, homostachydrine, unlike its lower homologue proline betaine (also known as stachydrine), showed a pattern of numerous ionic fragments that allowed unambiguous identification of the compound. For the quantitation in the plant sources, high sensitivity and specificity were achieved by monitoring the transition (158 → 72), which is absent in the fragmentation patterns of other major osmolytes commonly used as markers for studies of abiotic stress. As for the metabolic origin of homostachydrine, the occurrence in citrus plants of pipecolic acid leads to the hypothesis that it could act as a homostachydrine precursor through direct methylation.

Estimating Bergamot Juice Adulteration of Lemon Juice by High-Performance Liquid Chromatography (HPLC) Analysis of Flavanone Glycosides

The chemical composition of 30 samples of juices obtained from bergamot (Citrus bergamia Risso and Poit.) fruits is reported and compared to the genuineness parameters adopted by Association of the Industry of Juice and Nectars (AIJN) for lemon juice. It was found that the compositional differences between the two juices are distinguishable, although with difficulty. However, these differences are not strong enough to detect the fraudulent addition of bergamot juice to lemon juice. Instead, we found the high-performance liquid chromatography (HPLC) analysis of the flavanones naringin, neohesperidin, and neoeriocitrin, which are present in bergamot juice and practically absent in the lemon juice, is a convenient way to detect and quantify the fraudulent addition of bergamot juice. The method has been validated by calculating the detection and quantification limits according to Eurachem procedures. Employing neoeriocitrin (detection limit = 0.7 mg/L) and naringin (detection limit = 1 mg/L) as markers, it is possible to detect the addition of bergamot juice to lemon juice at the 1% level. When using neohesperidin as a marker (detection limit = 1 mg/L), the minimal percentage of detectable addition of bergamot juice was about 2%. Finally, it is reported that the pattern of flavonoid content of the bergamot juice is similar to those of chinotto (Citrus myrtifolia Raf) and bitter orange (Citrus aurantium L.) juices and that it is possible to distinguish the three kinds of juices by HPLC analysis.

Proline Derivatives in Fruits of Bergamot (Citrus bergamia Risso et Poit): Presence of N-Methyl-l-proline and 4-Hydroxy-l-Prolinebetaine

The content of proline and various compounds deriving from its metabolism (4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine) was determined in fruits and seeds of Bergamot (Citrus bergamia Risso et Poit), growing in the Calabria region (South Italy). A HPLC-ESI-tandem mass spectrometry method, which allowed rapid determination of L-proline, 4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine in juice and extracts of bergamot fruit with minimum sample preparation and short analysis time (about 10 min), is presented. Proline and 4-hydroxy-L-proline levels in the samples were also determined by HPLC analysis with fluorescence detection and the results compared to those obtained with HPLC-ESI-tandem mass spectrometry. For the first time, the presence of N-methyl-L-proline and 4-hydroxy-L-prolinebetaine in the fruits of a plant of the Citrus genus is reported.

Where Does Nε-Trimethyllysine for the Carnitine Biosynthesis in Mammals Come from?

Nε-trimethyllysine (TML) is a non-protein amino acid which takes part in the biosynthesis of carnitine. In mammals, the breakdown of endogenous proteins containing TML residues is recognized as starting point for the carnitine biosynthesis. Here, we document that one of the main sources of TML could be the vegetables which represent an important part of daily alimentation for most mammals. A HPLC-ESI-MS/MS method, which we previously developed for the analysis of NG-methylarginines, was utilized to quantitate TML in numerous vegetables. We report that TML, believed to be rather rare in plants as free amino acid, is, instead, ubiquitous in them and at not negligible levels. The occurrence of TML has been also confirmed in some vegetables by a HPLC method with fluorescence detection. Our results establish that TML can be introduced as free amino acid in conspicuous amounts from vegetables. The current opinion is that mammals utilize the breakdown of their endogenous proteins containing TML residues as starting point for carnitine biosynthesis. However, our finding raises the question of whether a tortuous and energy expensive route as the one of TML formation from the breakdown of endogenous proteins is really preferred when the substance is so easily available in vegetable foods. On the basis of this result, it must be taken into account that in mammals TML might be mainly introduced by diet. However, when the alimentary intake becomes insufficient, as during starvation, it might be supplied by endogenous protein breakdown.