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Every year tons of pistachio hulls are separated and eliminated, as waste products, from pistachio seeds. In this study the hulls of ripe pistachios were extracted with two organic solvents (ethanol and methanol) and characterized for phenolic composition, antioxidant power and cytoprotective activity. RP-HPLC-DAD-FLU separation enabled us to identify 20 derivatives, including and by far the most abundant gallic acid, 4-hydroxybenzoic acid, protocatechuic acid, naringin, eriodictyol-7-O-glucoside, isorhamnetin-7-O-glucoside, quercetin-3-O-rutinoside, isorhamnetin-3-O-glucoside and catechin. Methanol extraction gave the highest yields for all classes of compounds and presented a higher scavenging activity in all the antioxidant assays performed. The same was found for cytoprotective activity on lymphocytes, lipid peroxidation and protein degradation. These findings highlight the strong antioxidant and cytoprotective activity of the extract components, and illustrate how a waste product can be used as a source of nutraceuticals to employ in manufacturing industry.

Sanguinello and Tarocco are the blood orange ( Citrus sinensis (L.) Osbeck) cultivars most diffused worldwide. Reversed phase liquid chromatography coupled with MS-MS analysis showed that these two varieties have a similar chromatographic pattern, characterised by the presence of C - and O -glycosyl flavonoids. Of the two, Sanguinello was found to be far richer in flavonoids than Tarocco. In the juices, twelve individual components were identified for the first time, namely, four C -glycosyl flavones (lucenin-2, vicenin-2, stellarin-2, lucenin-2 4′-methyl ether and scoparin), three flavonol derivatives (quercetin-3- O -(2-rhamnosyl)-rutinoside, quercetin-3- O -hexoside, quercetin 3-hydroxy-3-methylglutaryl-glycoside), an O -triglycosyl flavanone (narirutin 4′- O -glucoside) and a flavone O -glycosides (chrysoeriol 7- O -neoesperidoside). Moreover, the influence of the identified C - and O -glycosyl flavonoids on the antioxidant and acetylcholinesterase activity of these juices has been evaluated.

RP-LC-DAD-ESI-MS-MS separation/identification protocol has been employed for the identification and characterisation of nine C- and O-glycosyl flavonoids in Moro (Citrus sinensis (L.) Osbeck) juice grown in Southern Italy. For the first time we reported the presence of five C-glycosyl flavones (lucenin-2, vicenin-2, stellarin-2, lucenin-2 4'-methyl ether and scoparin), a 3-hydroxy-3-methylglutaryl glycosyl flavonol (3-hydroxy-3-methylglutaryl glycosyl quercetin) and a flavone O-glycosides (chrysoeriol 7-O-neoesperidoside). Moreover, the influence of the identified C- and O-glycosyl flavonoids on the total antioxidant activity of crude juice has been evaluated on the basis of its ability to scavenge DPPH•, OH• and ABTS•+ radicals and to reduce iron.

A separation/identification protocol based on RP-LC-DAD-ESI-MS-MS has been employed for the characterisation of the flavonoid fraction of the juice from tangelos (Citrus reticulata×Citrus paradisi) grown in Southern Italy. Eleven compounds were identified in a single chromatographic course. Of these, two C-glycosyl flavones (lucenin-2 and vicenin-2) and an O-triglycosyl flavanone (narirutin 4'-O-glucoside) were identified for the first time. Fruit juice antioxidant activity was evaluated on the basis of its ability to scavenge DPPH, O2(-), OH and ABTS(+) radicals, and to reduce iron (FRAP). Moreover, the influence of the identified polymethoxylated, C- and O-glycosyl flavonoids on the total antioxidant activity has been elucidated. We also checked the antimicrobial activity of a broad fraction, containing all the detected flavonoids obtained by preparative HPLC, in terms of MICs for Staphylococcus aureus.

The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation–deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine–haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO2 thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane. Furthermore caffeine destabilizes the haeme–protein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silico docking and molecular dynamics studies and by what we may call the “caspase silence”; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing power.

Citrus myrtifolia Raf. (chinotto) flavedo, albedo and carpel membranes from green and ripe fruits, as well as seeds and leaves, were investigated for the first time for their flavonoid and furocoumarin composition. Twenty-three unique compounds distributed in the different fruit/plant parts were identified and quantified. All samples analysed contained flavanone O -glycosides, flavone C - and O -glycosides and furocoumarins; flavedos and albedos also contained significant amounts of polymethoxyflavones. Flavedo and albedo extracts were found to be richest in flavonoids and furocoumarins, containing up to 1.95 g/kg fresh weight. Flavedo, albedo and carpel membranes from ripe fruits were found to be richer than the corresponding tissues from unripe fruits. The remarkable radical-scavenging activity of all the extracts was tested by DPPH • , ABTS •+ and FRAP methods, revealing that (i) they were particularly efficient in quenching ABTS •+ radical cations (up to 9.8 mM Trolox equivalents), and (ii) flavedo and albedo extracts, on average, showed the highest antioxidant capacity.

Palytoxin (PTX) is classified as one of the most powerful marine biotoxins (of high molecular weight and no protein origin) because it is able to interact strongly with important cellular structures influencing their function in different biological processes. This study of the effects of PTX on red blood cells (RBC) extends the knowledge about its toxicity, which concerns not only the well-known action on Na(+)/K(+)-ATPase but also band 3 protein (B3 or AE1), the role of which is essential for anion transport and for the structure, function, and metabolic integrity of the erythrocyte. The effects of PTX on RBC can be summarized as follows: it alters the anionic flux and seriously compromises not only CO(2) transport but also the metabolic modulation centered on the oxy-deoxy cycle of hemoglobin; it stabilizes the plasma membrane by preventing lipid peroxidation; and its effect does not lead to activation of caspases 3 and 8. From what is reported in steps 2 and 3, and on the basis of the results obtained on hemolysis, methemoglobin levels, and phosphatase activity, an increase of the reducing power of the erythrocytes (RBC) in the presence of PTX clearly emerges. The results have enabled us to outline some metabolic adaptations induced in the RBC by PTX.

The qualitative and quantitative flavonoid and furocoumarin composition of crude sour orange (Citrus aurantium L.) juice was determined via reverse-phase LC–DAD–ESI–MS–MS analysis. In a single course, a comprehensive chromatographic profile of 14 compounds (furocoumarins, C- and O-glycosyl flavonoids) was obtained. Seven flavonoids and a furocoumarin were identified for the first time in sour orange juice: three C-glucosides (lucenin-2, vicenin-2 and lucenin-2, 4′-methyl ether), two O-glycosides (rhoifolin 4′-glucoside and narirutin 4′-glucoside), two 3-hydroxy-3-methylglutaryl flavanone glycosides (melitidin and brutieridin) and a furocoumarin (epoxybergamottin). Furthermore, the antioxidant activity of sour orange juice was assessed by means of ABTS + and DPPH radical scavenging assays.

The antioxidative activity of some natural flavonoids was analyzed against the stable free radical 2,2-diphenyl-1-picryhydrazyl. The results indicate that the scavenging power of the tested flavonols is higher than that of the synthetic antioxidants butylated hydroxyanisole and butylated hydroxytoluene; instead, the flavanones show little activity, as indicated by efficient concentration (EC50) values. Flavonoid autoxidation and interaction with Fe2+ and hydrogen peroxide were tested using erythrocyte membranes as a model. The results show that some compounds, like hesperetin, evidence a pro-oxidant activity higher than the ascorbic acid/iron reference system. The compounds with strong oxidative capability do not only influence cellular redox balance but also activate caspase-3, producing lactate dehydrogenase release and enhancing anionic exchange at the level of band 3 protein.

The anti-proliferative activity of hesperetin, hesperidin, neohesperidin and rutin was evaluated on human hepatoma cell lines (Hep G2) and correlated to their antioxidant activity. The results obtained showed strong anti-proliferative effects of hesperidin and neohesperidin, considerably higher than the other two additives. Hesperetin induced caspase-3 activation, release of LDH and endogenous accumulation of putrescine. Cell cycle distribution seems to indicate that the inhibitory effects of polyphenols on cell growth could be due to G0/G1 block, and activation of apoptotic pathway in the presence of hesperetin. Our results underline also that the glycone forms show reduced scavenging activity against DPPH, but present a remarkable inhibition of cell proliferation and low cytotoxicity.

HPLC separation of flavonoids and furocoumarins in the crude juices of three cultivars of Citrus bergamia Risso ("Castagnaro", "Fantastico", and "Femminello") was carried out on a C18 reversed phase column. The analysis was performed in a single run using a DAD detector coupled with an ESI-MS-MS source. Two furocoumarins (bergapten and bergamottin) were detected and quantified simultaneously with the sixteen flavonoid components previously found in industrial bergamot juice. Full characterization of the furocoumarins was performed by (1)H NMR analysis on samples separated by means of preparative HPLC. The free-radical scavenging ability of cultivar juices was assessed by using DPPH radical. The data presented show that the "Femminello" cultivar, even though it is the least common of the three, is by far the richest in health-promoting bioactive compounds (both flavonoids and furocoumarins). Given the range of applications of furocoumarins, the preparative separation described herein is proposed as a simple and rapid method to obtain this class of compounds in good yield from crude juice.

6,8-Di-C-glucopyranosylapigenin and 6,8-di-C-glucopyranosyldiosmetin were detected by HPLC–DAD–ESI–MS–MS in a variety of Southern Italian Citrus juices (orange, lemon, bergamot, citron, mandarin, clementine). In some juices, the amounts of C-glycosides detected were significant. 6,8-Di-C-glucopyranosylapigenin is characteristic of orange juice, while 6,8-di-C-glucopyranosyldiosmetin is the most important C-glycoside in lemon and citron juice. In bergamot juice the concentrations of 6,8-di-C-glucopyranosylapigenin and 6,8-di-C-glucopyranosyldiosmetin are similar. Clementine juice is distinctive as the amounts of both C-glycosides present are negligible.

Ornithine carbamoyltransferase (OCT) from spinach (Spinacea oleracea L.) was purified to homogeneity and studied for some kinetic and structural properties. The enzyme showed a specific activity of 436 U mg−1, its molecular mass was approximately 118 kDa as estimated by Sephacryl S-200 gel filtration chromatography, the purified protein ran as a single band of 38 kDa in sodium dodecyl sulfate-polyacryamide gel electrophoresis. The enzyme catalyses an ordered bi-bi-sequential reaction in which carbamoyl phosphate binds first, followed by L-ornithine; L-citrulline leaves first, followed by phosphate. The Michaelis constant was 0.19 mM for L-ornithine and 13.1 µM for carbamoyl phosphate; the dissociation constant for the enzyme and carbamoyl phosphate complex was of 19 µM. The Km of the reaction decreases from pH 6.0 to pH 10.4. The enzyme is heat-labile, but it was protected from thermal inactivation by substrates; more by ornithine alone than by two substrates acting together.

Ornithine carbamoyltransferase folding/unfolding is a complex and not completely understood process. Our experimental results suggest that ornithine carbamoyltransferase interacts in a completely different way with urea and guanidine hydrochloride. In fact, we noticed that, increasing concentration from 0.0 to 8.0 M of the two additives, the enzyme follows a simple one-step transition mechanism in the presence of guanidine hydrochloride, with two macroscopic states (the native and the denatured one) significantly populated, whereas in the presence of urea a lot of different protein states can be detected and analyzed. Circular dichroism and UV-visible spectroscopy reveal a similar mechanism of perturbation at high temperature, with opening of hydrophobic core and a significant loss in α-helix structure in the presence of guanidine hydrochloride that cannot be found in the presence of urea.

Carbamazepine (CBZ) is an iminostilbene derivative commonly used for treatment of neuralgic pain and bipolar affective disorders. CBZ blood levels of treated patients are within the range of micromolar concentrations and therefore, significant interactions of this drug with erythrocytes are very likely. Moreover, the lipid domains of the cell membrane are believed to be one of the sites where iminostilbene derivatives exert their effects. The present study aimed to deeply characterize CBZ effects on erythrocytes, in order to identify extra and/or cytosolic cell targets. Our results indicate that erythrocyte morphological changes promoted by the drug, may be triggered by an alteration in band 3 functionality i.e. at the level of anionic flux. In addition, from a metabolic point of view this perturbation could be considered, at least in part, as a beneficial event because it could favour the CO2 elimination. Since lipid peroxidation, superoxide and free radical scavenging activities, caspase 3 activity and hemoglobin (Hb) functionality were not modified within the CBZ treated red blood cell (RBC), band 3 protein (B3) may well be a specific membrane target for CBZ and responsible for CBZ-induced toxic effects in erythrocytes. However some beneficial effects of this drug have been evidenced; among them an increased release of ATP and nitric oxide (NO) derived metabolites from erythrocytes to lumen, leading to an increased NO pool in the vasculature. In conclusion, these results indicate that CBZ, though considered responsible for toxic effects on erythrocytes, can also exhibit effects that at least in some conditions may be seen as beneficial.

The flavonoid composition of crude Citrus limetta Risso (limetta, Mediterranean sweet lemon) juice was elucidated by means of reverse-phase HPLC–DAD–ESI-MS/MS analysis. In a single course, eight compounds (C- and O-glycosyl flavonoids) were identified, six of which were found for the first time in limetta juice: four C-glucosyl flavones, namely vicenin-2, lucenin-2 4′-methyl ether, orientin 4′-methyl ether and scoparin; the O-glycosyl flavone, rhoifolin; and the O-glycosyl flavanone, eriocitrin. In addition, ABTS + and DPPH radical quenching and FRAP assays demonstrate that limetta juice possesses remarkable antioxidant activity.

Cherimoya is a tropical and subtropical native exotic fruit which has become an important crop due to its tasty flavor, high pulp content, nutritional value and potential use in folk medicine. In this study the pulp of ripe fruits were extracted using three organic solvents (ethanol, methanol and dimethyl formammide) and analyzed for the total phenols, antioxidants power and cytoprotective activity on lymphocyte tert-butyl hydroperoxide treated. Also, potential antiperoxidative activity of each extract has been performed. Organic extracts from cherimoya pulp had powerful antioxidant and ferric reducing power activities and, among the extracts, the dimethyl formammide one showed the highest scavenging activity towards DPPH , ABTS +, O2 − radical and FRAP assay, while the ethanol one showed the highest activity against lipid peroxidation induced by tert-butyl hydroperoxide. Extract protective activities were also evaluated in isolated human peripheral blood lymphocytes exposed to 250 μM tert-butyl hydroperoxide. The results showed that all the extracts could significantly enhance cell survival and clearly decrease the release of lactate dehydrogenase, with data almost superimposable for all the tested samples. Moreover RP-HPLC-DAD-ITMS analysis reveals the presence of flavanols and procyanidins (dimers and trimers). These results highlight cherimoya extracts' strong antioxidant properties, remarkable decrease of lipid peroxidation and cytoprotective effects against strong oxidant, supporting their suitable employment as bioactive elements in cosmetic, pharmaceutical and food processing industries.

The flavonoids composition of kumquat (Fortunella japonica) crude juice, obtained from uripe and ripe fruits, were investigated by reverse-phase LC-DAD-ESI-ITMS analysis. Thirteen compounds (C- and O-glycosyl flavonoids) were identified. Acacetin 3,6-di-C-glucoside (1), vicenin-2 (2), lucenin-2 4′-methyl ether (3) narirutin 4′-O-glucoside (4) and apigenin 8-C-neohesperidoside (5) were identified for the first time in kumquat juice. Furthermore, we report on the remarkable antioxidant properties of crude juice, of selected flavonoids fractions and of the main component, phloretin 3′,5′-di-C-glucoside (6).

Resveratrol, an important phytoalexine found in many plants, has been shown to be significantly effective in the treatment of several pathological conditions such as cancer, coronary heart disease and osteoarthritis. This study focuses on the effects of this drug on human red blood cells. In particular, we have examined the influence of resveratrol on Band 3, the anion exchanger protein, and hemoglobin as a function of the oxygenation-deoxygenation cycle. Moreover, special attention has been given to the metabolic changes imposed by caspase 3 activation. Resveratrol has proved to lower superoxide production, thereby decreasing heme-iron oxidation and saving the reducing power required for met-hemoglobin reduction. Oxygen binding experiments showed that resveratrol interacts with hemoglobin, shifting the T→R conformational transition towards the higher-affinity R state. This might contribute to altering the metabolic balance of the cell through an intensification of the pentose phosphate pathway. Moreover, at high oxygenation levels of the erythrocytic hemoglobin, resveratrol induces a significant activation of caspase 3, the action of which on Band 3 has a strong impact on cellular metabolism and anion transport.

The qualitative and quantitative compositions of chinotto juice in two different maturation periods were determined via chromatographic separation of extracted aliquots of juice of Citrus x myrtifolia Raf. by using reverse-phase LC-DAD-ESI-MS-MS. This provides a comprehensive chromatographic evaluation of 11 compounds (furanocoumarins and flavonoids C- and O-glycosides). Five flavonoids and two furanocoumarins were identified for the first time in chinotto juice: two C-glucosides (vicenin-2 and lucenin-2 4'-methyl ether), two O-glycosides (narirutin and rhoifolin), and a 3-hydroxy-3-methylglutaryl flavanone (brutieridin). Bergapten and epoxybergamottin were the primary furanocoumarins found. Overall, the juice from immature chinotto fruits is richer in bioactive compounds than that obtained from ripe fruits. The free radical and superoxide anion scavenging activities of juice from both green and ripe fruits were assessed, and results showed that the former is much more efficient in scavenging radical and superoxide species than the latter.

During our experimental work, aggregation of bovine serum albumin was obtained incubating the protein solution at 60 degrees C to investigate temperature-induced secondary structure, conformation changes and anti-aggregative activity of trehalose. IR-measurements suggested that in the presence of 1.0M of trehalose there is a little increase in short segment connecting alpha-helical and a clearly decrease in the loss of alpha-helix structure and in the formation of intermolecular and antiparellel beta-sheet up to 78 and 55%, respectively. Useful information also arose following the temperature evolution of Amide I' band profile in the range of temperature between 25 and 90 degrees C in absence or in presence of 1.0M trehalose. Complementary information is obtained by electrophoresis, circular dichroism, fluorescence spectroscopy, titration of SH groups and light scattering measurements. Results encouraged biotechnology and pharmaceutical application of the disaccharide and provided evidence for its utilization in degenerative diseases evolving via aggregation process.

In the present article the influence of salts and additives, such as trehalose, NaCl, ornithine, sodium phosphate and ammonium sulphate, on ornithine carbamoyltransferase (OCTase) is investigated in order to study the OCTase stabilization process as a function of solutes and to point out the fundamental role played by an enhancement of hydrophobic interactions. The synergic use of different techniques, such as neutron spectroscopy, UV-vis spectroscopy, activity and thermal measurements, allows to highlight the cosolute capability to avoid thermal inactivation, to induce important changes in secondary and tertiary enzyme structure and to stabilize biological macromolecules.

Thermodynamic, circular dichroism (CD), and activity measurements have been used to characterize the different conformational states and the effects of NaCl concentrations (0.0–3.0 M) on thermal unfolding of ornithine carbamoyltransferase (OCT) from Alopias vulpinus . Furthermore conformational changes in whole enzyme structure have been monitored by titration of SH-groups. OCT unfolding process follows an irreversible two-state mechanism with a first-order kinetic of denaturation, without breaking-point. NaCl shows very little stabilization effects at low concentration and its action become very important over 1.5 M concentration. The presence of 3.0 M NaCl completely avoids OCT unfolding at 60, 64 and 66 °C. Kinetic and thermodynamic parameters are strongly influenced by the presence of high NaCl concentration. Our experiments showed that NaCl stabilization process involved changes in preferential binding, in electrostatic and van der Waals interactions and exposure of buried site and SH-groups. During thermal denaturation, UV–vis and CD spectroscopy show that high salts concentration preserves OCT activity, avoiding exposure of hydrophobic site and destruction of secondary and tertiary structure elements.

During vertebrate evolution, structural changes in red blood cells (RBC) and hemoglobin (Hb), have probably resulted in the importance of blood carbon dioxide transport. The chloride/bicarbonate exchange across the RBC membrane, which is an integral part of the blood CO2 transport process in vertebrates, has been examined on two different species of teleost fish, Euthynnus alletteratus and Thunnus thynnus, at several oxygenation states of erythrocyte HOS (high-oxygenation state, about 90 % of saturation) and LOS (low-oxygenation state, about 15 % of saturation). The results were compared with those observed in human RBC under the same experimental conditions and with the chicken (Gallus gallus) erythrocytes, which have particular modifications at the N-terminus of the band 3 protein (B3). In fish the kinetic measurements have shown a different anion transport in several oxygenation states of erythrocytes, indicating that also at lower levels of vertebrate evolution there exists a modulation of the anionic flow affected by oxygen. The functional correlation of anion transport to changes of parts of the hemoglobin sequence responsible for alterations in the interactions with the cytoplasmic domain of band 3 protein (cdb3) allowed us to suggest a hypothesis about fish physiology. The highest values of kinetic measurements observed in fish have been attributed to the metabolic need of the RBC in response to the removal of CO2 that in teleosts is also of endogenous origin.

Cultivars and consumption typologies of some Allium species can significantly vary from a chemical point of view and even small differences can be important for their characterization and differentiation. Bulbs of three varieties and four consumption typologies of onion (Allium cepa L.) and two varieties of shallot (Allium ascalonicum Hort.) were subjected to HPLC–DAD–ESI-MS–MS analysis. Seven flavonol glucosides were identified in all the samples, two of which, quercetin 3,4′-diglucoside and quercetin 4′-glucoside, represent about the 90% of the overall contents. Cultivars and consumption typologies of the Allium species under study show significant differences in flavonol contents, from the very low quantity of antioxidant compounds in white onion, about 7 mg/kg against 600–700 mg/kg that were found in red and gold varieties, to the enormous content of flavonols that are present in onions of prompt consumption, where quercetin 4′-glucoside exceeds 1 g/kg and quercetin 3-glucoside is present in a ratio higher then 10:1 with respect to its value in the other onion typologies. Shallots are very rich in the two major flavonols.

In this work, we investigate the effects of different cosolutes on ornithine carbamoyltransferase in order to study the role of water in stabilization processes as a function of solutes and to point out the fundamental role played by an enhancement of hydrophobic interactions. It is well known that the cosolutes can affect the solvent properties and therefore influence the environment in which biostructures are immersed. Structure-breakers or “chaotrope” solutes do not stabilize biomolecules, whereas structure-makers or “kosmotrope” solutes are capable to create strong bonds with water, so creating good conditions for avoiding denaturation processes. The effect of different additives and temperature on ornithine carbamoyltransferase has been carried out using techniques such as the measurement of the enzymatic activity and UV–VIS spectroscopy.

The present paper shows a systematic dynamic and kinetic study on l -ascorbic acid and its degradation at high temperature. The neutron scattering study allows, through the behavior of quasi-elastic neutron scattering (QENS) spectra, to characterize the diffusive dynamics of l -ascorbic acid in water mixtures. Ascorbic acid undergoes degradation process at high temperature, but the presence of trehalose in solution markedly avoids ascorbic acid loss enhancing its t1/2 (half life time), as determined by high performance liquid chromatography (HPLC).

In Pontonia pinnophylax (Otto), a crustacean decapod inhabiting the mantle cavity of Pinna nobilis L. (Bivalvia: Pteriomorpha), the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, and their electrophoretic patterns, were compared in relation to heat and urea inactivation. Activity was higher in LDH than in MDH, and the electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH. Heat incubation reduced both enzymatic activities, but more MDH. Also all isozymes showed different heat sensitivity, with anodic forms more heat-resistant than cathodic ones, either in LDH as in MDH. Urea treatment caused also a higher inactivation of the most cathodic isozymes, but MDH appeared more resistant than LDH at 2 M urea. The high polymorphism of these enzymes suggests an adaptation of Pontonia metabolism to hypoxic conditions; moreover, the different isozyme stability grade should be functional to contrast environmental variability.

A comprehensive profile of flavonoids in bergamot juice was obtained by a single DAD-ESI-LC-MS-MS course. Eight flavonoids were found for the first time, five of these are C-glucosides (lucenin-2, stellarin-2, isovitexin, scoparin, and orientin 4'-methyl ether), and three are O-glycosides (rhoifolin 4'-O-glucoside, chrysoeriol 7-O-neohesperidoside-4'-O-glucoside, and chrysoeriol 7-O-neohesperidoside). A method is proposed to differentiate chrysoeriol and diosmetin derivatives, which are often indistinguishable by LC-MS-MS. In-depth knowledge of the flavonoid content is the starting point for bergamot juice exploitation in food industry applications.

High-performance liquid chromatography-diode array detector (HPLC-DAD) coupled with electron spray mass spectrometry (ESI-MS-MS) was used to determine the flavonol profile in southern Italian red onions (Allium cepa L.). This on-line technique allowed the identification of seven flavonols in southern Italian red onion, quercetin 4'-glucoside and quercetin 3,4'-diglucoside being the most abundant components. Five minor flavonols have been recognized, offering a characteristic profile of such compounds in red onions under study. Quercetin 3-glucoside, quercetin 7,4'-diglucoside, quercetin 3,7,4'-triglucoside, and isorhamnetin 4'-glucoside have been previously reported as minor flavonoid components in Allium cepa, while isorhamnetin 3,4'-diglucoside was previously found in Allium ascalonicum. Traces of isorhamnetin 3-glucoside and free quercetin were also detected.

High-performance liquid chromatography-diode array detector (HPLC-DAD) coupled with electronspray mass spectrometry (ESI-MS) was used to detect the flavonoid profile in lemon juices obtained from the main Sicilian cultivars (Femminello comune, Monachello, and Interdonato). Significant amounts of an unusual constituent were found in the lemon juice of the above-mentioned cultivars together with eriocitrin, hesperidin, and diosmin. Following purification by preparative HPLC, the structure was assigned as 6,8-di-C-glucopyranosyldiosmetin by means of DAD-UV, ESI-MS-MS, and (1)H NMR analysis. Three other minor components were also detected. One of these presented a flavone nature, and spectral data and literature references both suggested a 6,8-di-C-glucopyranosylapigenin structure. The different contents of eriocitrin, hesperidin, diosmin, and above minor components in the cultivars allow juices to be readily differentiated.

Flavanone glycosides (FGs) and polymethoxylated flavones (PMFs) have been studied in pigmented orange (Citrus sinensis) juices and second-pressure extracts (SPEs) by high-performance liquid chromatography and diode array detector. Detection was performed simultaneously at two different wavelengths: 278 nm (for determination of FGs) and 325 nm (for determination of PMFs). Qualitative distribution patterns of FGs and PMFs in juices and SPEs were similar, although the quantitative results are quite different. An increased narirutin/hesperidin ratio after centrifugation and the presence of high amounts of PMFs in SPEs, which remain unchanged after centrifugation, were observed. Therefore, a simple and affordable procedure to distinguish an orange juice from SPEs was proposed.

Recognition of adulteration by other oils via direct analysis of the minor components (“sterol fraction”) is shown for olive oil. 10 % of various oils were admixed, the free alcohols silylated and the minor components analyzed by on-line coupled LC-GC-FID. For most oils, even smaller additions can be recognized. Admixed oils can no longer be determined, however, if their minor components have been removed by strong raffination. Bleaching of rapeseed oil with 7% of earth at 180 °C, in fact, completely removed free and esterified sterols.