First characterization of Pompia intrea candied fruit: The headspace chemical profile, polar extract composition and its biological activities.

Extracts obtained from Pompia intrea (PI), a traditional candied fruit whose main ingredient is the pompia fruit (Citrus monstruosa L.) were evaluated for the first time. Volatile compounds were evaluated by headspace solid-phase microextraction (HS-SPME) followed by GC-FID/MS analyses. Polar compounds were identified by high-performance liquid chromatography coupled with electrospray mass spectrometry LC-ESI-Orbitrap-MS/MS and quantified by liquid chromatography coupled with ultraviolet/visible detection (LC-DAD). The antioxidant activity of these extracts was tested using the FRAP, CUPRAC, ABTS•+ and DPPH● assays. Moreover, their ability to protect intestinal cells against lipid peroxidation was studied. The HS-SPME GC-FID/MS confirmed the presence of typical molecules originating from the fruit (mainly terpenes, but particularly limonene). The LC-DAD and LC-ESI-(HR)MSn profiles showed high levels of neohesperidin (45.7 ±â€¯11.1 mg/L) and 5-(hydroxymethyl)furfural (40.8 ±â€¯23.5 mg/L). The results showed that the PI extracts contained high levels of total phenols and exhibited considerable antioxidant activity, which was significantly correlated to the presence of specific compounds such as neoeriocitrin and neohesperidin. Furthermore, pretreatments with different concentrations of PI extracts preserved enterocytes from oxidative damage by scavenging reactive oxygen species, thus counteracting lipid peroxidation. This study suggests that consumption of PI could provide intake of compounds with ascertained biological activity.

Derangement of intestinal epithelial cell monolayer by dietary cholesterol oxidation products.

The emerging role of the diet in the incidence of intestinal inflammatory diseases has stimulated research on the influence of eating habits with pro-inflammatory properties in inducing epithelial barrier disturbance. Cholesterol oxidation products, namely oxysterols, have been shown to promote and sustain oxidative/inflammatory reactions in human digestive tract. This work investigated in an in vitro model the potential ability of a combination of dietary oxysterols representative of a hyper-cholesterol diet to induce the loss of intestinal epithelial layer integrity. The components of the experimental mixture were the main oxysterols stemming from heat-induced cholesterol auto-oxidation, namely 7-ketocholesterol, 5α,6α-and 5ß,6ß-epoxycholesterol, 7α- and 7ß-hydroxycholesterol. These compounds added to monolayers of differentiated CaCo-2 cells in combination or singularly, caused a time-dependent induction of matrix metalloproteinases (MMP)-2 and -9, also known as gelatinases. The hyperactivation of MMP-2 and -9 was found to be associated with decreased levels of the tight junctions zonula occludens-1 (ZO-1), occludin and Junction Adhesion Molecule-A (JAM-A). Together with such a protein loss, particularly evident for ZO-1, a net perturbation of spatial localization of the three tight junctions was observed. Cell monolayer pre-treatment with the selective inhibitor of MMPs ARP100 or polyphenol (-)-epicathechin, previously shown to inhibit NADPH oxidase in the same model system, demonstrated that the decrease of the three tight junction proteins was mainly a consequence of MMPs induction, which was in turn dependent on the pro-oxidant property of the oxysterols investigated. Although further investigation on oxysterols intestinal layer damage mechanism is to be carried on, the consequent - but incomplete - prevention of oxysterols-dependent TJs alteration due to MMPs inhibition, avoided the loss of scaffold protein ZO-1, with possible significant recovery of intestinal monolayer integrity.

New insights into the antioxidant activity and cytotoxicity of arzanol and effect of methylation on its biological properties.

The heterodimeric phloroglucinyl pyrone arzanol (Arz) has raised considerable interest because of its antiviral, anti-inflammatory, and antioxidant activity. We have investigated the effect of methylation of the pyrone moiety on the antioxidant activity and cytotoxicity of Arz. This manoeuvre, that left the polyphenolic moiety unscathed, was nevertheless detrimental for antioxidant activity in both the cholesterol thermal degradation- and the Cu2+-induced liposome oxidation assays, providing evidence of structure-activity relationships that go beyond the preservation of the polyphenolic pharmacophore. The antioxidant activity of Arz was retained also in the Fe-NTA model of in vivo oxidative stress, with protective effect on the oxidative degradation of plasmatic lipids, unsaturated fatty acids and cholesterol. Both Arz and methylarzanol (Me-Arz) were devoid of toxic effect on colonic differentiated Caco-2 cells up to 100µM, but significantly reduced cancer Caco-2 cell viability at lower dosages. Arz could also selectively reduce viability of other cancer cell lines, [murine melanoma cells (B16F10 cells), human cervical carcinoma cells (HeLa cells)], suggesting that it can act as a selective modulator of cell processes typical of cancer cells. Taken together, our results qualify Arz as a lead structure for further in vivo investigation of its pharmacological potential.

Chemical composition of Lycium europaeum fruit oil obtained by supercritical CO2 extraction and evaluation of its antioxidant activity, cytotoxicity and cell absorption.

We studied the total phenols and flavonoids, liposoluble antioxidants, fatty acid and triacylglycerol profiles, and oxidative status of oil obtained from Lycium europaeum fruits following supercritical CO2 extraction (at 30MPa and 40°C). Linoleic (52%), palmitic (18%), oleic (13%), and α-linolenic (6%) were the main oil fatty acids, while trilinolein and palmitodilinolein/oleodilinolein represented the main triacylglycerols. The oil was characterized by high levels of all-trans-zeaxanthin and all-trans-ß-carotene (755 and 332µg/g of oil, respectively), α-tocopherol (308µg/g of oil), total phenols (13.6mg gallic acid equivalents/g of oil), and total flavonoids (6.8mg quercetin equivalents/g of oil). The oil showed radical scavenging activities (ABTS and DPPH assays) and inhibited Caco-2 cell growth. Moreover, the incubation of differentiated Caco-2 cells with a non-toxic oil concentration (100µg/mL) induced a significant intracellular accumulation of essential fatty acids. The results qualify L. europaeum oil as a potential source for food/pharmaceutical applications.

Antioxidant Effect of Natural Table Olives Phenolic Extract Against Oxidative Stress and Membrane Damage in Enterocyte-Like Cells.

The phenolic fraction of a naturally fermented cultivar of table olives, "Tonda di Cagliari," was investigated for the ability to protect Caco-2 cells against oxidative stress and membrane damage induced by tert-butyl hydroperoxyde (TBH). TBH exposure resulted in an alteration of cellular redox status, with an increase in reactive oxygen species (ROS) and a decrease in reduced glutathione (GSH) level. A loss of the epithelial integrity, as indicated by the decrease of the transepithelial electrical resistance value, was also observed over time, together with an intense lipid peroxidation process. The olives phenolic extract significantly counteracted ROS generation and subsequent alteration of monolayer integrity and membrane oxidative damage. The protective action of the extract is likely due to the scavenging ability of its main components, as hydroxytyrosol, oleuropein, and verbascoside among the secoiridoids and derivatives. Since olives phenolic compounds concentrate in the intestinal lumen, they may be a useful tool in the prevention of intestinal disorders related to oxidative damage.

Extra virgin olive oil phenolic extracts counteract the pro-oxidant effect of dietary oxidized lipids in human intestinal cells.

The phenolic fraction of extra virgin olive oil (EVOO) concentrates before absorption in the intestinal lumen, where it may contribute to the modulation of enterocytes response to oxidative and inflammatory stimuli. We evaluated the ability of two monovarietal EVOOs phenolic extracts, Bosana and Nera di Gonnos/Tonda di Cagliari, typical and widespread varieties in Sardinia (Italy), to counteract in enterocytes like Caco-2 cells the pro-oxidant action of oxidized lipids, tert-butyl hydroperoxide (TBH) or a mixture of oxysterols of dietary origin. We confirmed that TBH treatment causes a significant increase of ROS production, GSH depletion, increase of MDA, fatty acids hydroperoxides and 7-ketocholesterol, and showed first evidence of oxidative imbalance and cell damage due to oxysterols exposure. Preincubation of cells with the phenolic extracts significantly attenuated oxidative modifications. Bosana extract showed the highest concentration of total phenols, mainly hydroxytyrosol and tyrosol, and was the most active in presence of TBH, where the free radical scavenging activity of these simple phenols seems to be a determining factor. The two extracts were equally effective, in spite of the different composition, in presence of oxysterols, where ROS production probably occurs according to different and more complex mechanisms.

Maltese mushroom (Cynomorium coccineum L.) as source of oil with potential anticancer activity.

The present study aimed to examine the potential anticancer properties of fixed oil obtained from Maltese mushroom (Cynomorium coccineum L.), an edible, non-photosynthetic plant, used in traditional medicine of Mediterranean countries to treat various ailments and as an emergency food during the famine. We investigated the effect of the oil, obtained from dried stems by supercritical fractioned extraction with CO2, on B16F10 melanoma and colon cancer Caco-2 cell viability and lipid profile. The oil, rich in essential fatty acids (18:3n-3 and 18:2n-6), showed a significant growth inhibitory effect on melanoma and colon cancer cells. The incubation (24 h) with non-toxic oil concentrations (25 and 50 µg/mL) induced in both cancer cell lines a significant accumulation of the fatty acids 18:3n-3 and 18:2n-6 and an increase of the cellular levels of eicosapentaenoic acid (20:5n-3) with anticancer activity. Moreover, the oil exhibited the ability to potentiate the growth inhibitory effect of the antitumor drug 5-fluorouracil in Caco-2 cells and to influence the melanin content in B16F10 cells. The results qualify C. coccineum as a resource of oil, with potential benefits in cancer prevention, for nutraceutical and pharmaceutical applications.

Potential anti-tumor effects of Mugil cephalus processed roe extracts on colon cancer cells.

The salted-semidried mullet ovary product, bottarga, is a Mediterranean food rich in n-3 PUFA EPA and DHA. We studied and compared the effects on cell viability, sensitivity to the anti-tumor drug 5-fluorouracil, and lipid composition, in colon cancer Caco-2 cells after 24 h incubation with oils and hydrophilic extracts obtained from two bottarga samples stored at different conditions. The cellular absorption of bottarga lipids was assessed in cancer cells by the evaluation of lipid accumulation in cytoplasmic lipid droplets by fluorescence microscopy. Bottarga oil showed a significant in vitro inhibitory effect on the growth of cancer Caco-2 cells and the ability to potentiate, at non-toxic concentration, the growth inhibitory effect of 5-fluorouracil. Moreover, bottarga oil induced in cancer Caco-2 cells marked changes in fatty acid composition, with a significant accumulation of the n-3 PUFA EPA and DHA, and cytoplasmic lipid droplet formation. Also bottarga hydrophilic extract, characterized by means of ¹H NMR spectroscopy, exhibited a reduction in cancer cell viability, without affecting cell lipid profile. Cell cholesterol levels were unmodified by all treatments. The results showed interesting anti-tumor properties of bottarga lipids, and qualify this fish product as a food with nutraceutical properties and potential benefits in colon cancer prevention.

Chemical composition and effect on intestinal Caco-2 cell viability and lipid profile of fixed oil from Cynomorium coccineum L.

Cynomorium coccineum L. is a non-photosynthetic plant, spread over Mediterranean countries, amply used in traditional medicine. We investigated the composition and effect on intestinal Caco-2 cell viability and lipid profile of fixed oil obtained from dried stems of the plant. Oil isolation has been performed by supercritical fractioned extraction with CO2. 13C NMR spectroscopy has been used to study the molecular composition of oil lipids; fatty acid composition was identified using GC and HPLC techniques. The fixed oil was composed mainly by triacylglycerols and derivates. The main fatty acids were 18:1 n-9 (38%), 18:2 n-6 (20%), 16:0 (15%), and 18:3 n-3 (10.8%). The oil showed a significant in vitro inhibitory effect on the growth of colon cancer undifferentiated Caco-2 cells. Moreover, cell viability, lipid composition, and lipid peroxidation were measured in intestinal epithelial cells (differentiated Caco-2 cells) after 24 h incubation with fixed oil. The oil did not show a toxic effect on colon epithelial cell viability but induced a significant change in fatty acid composition, with a significant accumulation of the essential fatty acids 18:3 n-3 and 18:2 n-6. The results showed remarkable biological activity of Maltese mushroom oil, and qualify it as a potential resource for food/pharmaceutical applications.

Extraction and separation of volatile and fixed oils from seeds of Myristica fragrans by supercritical CO2: chemical composition and cytotoxic activity on Caco-2 cancer cells.

UNLABELLED: Isolation of volatile and fixed oils from nutmeg have been obtained by supercritical fractioned extraction with carbon dioxide. Extraction experiments were carried out at pressures of 90 and 250 bar and temperature of 40 °C. The extraction step performed at 90 bar produced a volatile fraction mainly formed by myristicin (32.8%), sabinene (16.1%), α-pinene (9.8%), ß-pinene (9.4%), ß-phellandrene (4.9%), safrole (4.1%) and terpinen-4-ol (3.6%). The oil yield relative to this step of the process was 1.4% by weight of the charge. The last extraction step at 250 bar produced a butter-like material (nutmeg butter). The yield of this step was 14.4% by weight. The most represented fatty acids of fixed oil from nutmeg were 14:0 (79.2%), 18:1 n-9 (7.4%) and 16:0 (6.1%), and in particular the unsaturated fatty acids 18:1 n-9 averaged 32.96 µg/mg of oil. The level of myristicin in the nutmeg essential and fixed oils was also directly quantified by reversed HPLC-DAD. Moreover, the essential oil obtained from nutmeg, as well as myristicin, showed a significant in vitro inhibitory effect on the growth of a colon cancer cell line (undifferentiated Caco-2 cells). PRACTICAL APPLICATION: In this study, the chemical characterization and the anticancer activity of nutmeg oils obtained by supercritical extraction with carbon dioxide were investigated. This is important for their potential application in food and pharmaceutical industries.

Wine extracts from Sardinian grape varieties attenuate membrane oxidative damage in Caco-2 cell monolayers.

One of the most important sites of polyphenol action seems to be in the gastrointestinal system before absorption. We investigated the ability of three wine phenolic extracts, obtained from grape varieties grown in Sardinia, Cannonau (red), Vermentino and Malvasia (white), to exert an antioxidant action against tert-butyl hydroperoxide (TBH)-induced oxidative damage to Caco-2 cell monolayers as a model system of the human intestine. TBH treatment caused the disruption of epithelial integrity, measured as transepithelial electrical resistance, and markers of the peroxidation process of membrane lipids, MDA, fatty acid hydroperoxides and 7-ketocholesterol. All wine extracts were able to counteract the oxidising action of TBH and, in spite of the differences in phenolic composition, exerted a comparable activity. Our findings point out a direct antioxidant action of the wine extracts on enterocytes exposed to oxidising species and further support the opinion that total phenolic content is not essential for antioxidant activity.

Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage.

Hydroxytyrosol (2-(3',4'-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3'-O-ß-d-glucuronide and 4'-O-ß-d-glucuronide derivatives and 2-(3',4'-dihydroxyphenyl)ethanol-1-O-ß-d-glucuronide, in comparison with the parent compound, to inhibit H(2)O(2) induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H(2)O(2) treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited.

Effect of aqueous and lipophilic mullet (Mugil cephalus) Bottarga extracts on the growth and lipid profile of intestinal Caco-2 cells.

The importance of n-3 polyunsaturated fatty acid (n-3 PUFA) intake has long been recognized in human nutrition. Although health benefits, n-3 PUFA are subject to rapid and/or extensive oxidation during processing and storage, resulting in potential alteration in nutritional composition and quality of food. Bottarga, a salted and semi-dried mullet ( Mugil cephalus ) ovary product, is proposed as an important source of n-3 PUFA, having high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we investigated the extent of lipid oxidation of grated bottarga samples during 7 months of storage at -20 °C and room temperature under light exposure. Cell viability, lipid composition, and lipid peroxidation were measured in intestinal differentiated Caco-2 cell monolayers after 6-48 h of incubation with lipid and hydrophilic extracts obtained from bottarga samples at different storage conditions. The storage of bottarga did not affect the n-3 PUFA level, but differences were observed in hydroperoxide levels in samples from different storage conditions. All tested bottarga extracts did not show a toxic effect on cell viability of differentiated Caco-2 cells. Epithelial cells incubated with bottarga oil had significant changes in fatty acid composition but not in cholesterol levels with an accumulation of EPA, DHA, and 22:5. Cell hydroperoxides were higher in treated cells, in relation to the oxidative status of bottarga oil. Moreover, the bottarga lipid extract showed an in vitro inhibitory effect on the growth of a colon cancer cell line (undifferentiated Caco-2 cells).

Antioxidant profile of strawberry tree honey and its marker homogentisic acid in several models of oxidative stress.

The antioxidant activity of several honeys was evaluated considering the different contribution of entire samples. The strawberry tree honey emerged as the richest in total phenols and the most active honey in the DPPH and FRAP tests, and could protect cholesterol against oxidative degradation (140°C). Homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA), the main phenolic compound from strawberry tree honey, showed interesting antioxidant and antiradical activities, and protective effect against thermal-cholesterol degradation, comparable to those of well known antioxidants. Moreover, the pre-treatment with HGA significantly preserved liposomes and LDL from Cu(2+)-induced oxidative damage at 37°C for 2h, inhibiting the reduction of polyunsaturated fatty acids and cholesterol and the increase of their oxidative products. This phenol had no toxic effect in human intestinal epithelial Caco-2 cells within the concentration range tested (5-1000µM). HGA was able to pass through the Caco-2 monolayers, the apparent permeability coefficients (Papp) in the apical-to-basolateral and basolateral-to-apical direction were 3.48±1.22×10(-6) and 2.18±0.34×10(-6)cm/s, respectively, suggesting a passive diffusion pathway as the dominating process. The results of the work qualify HGA as natural antioxidant, able to exert a significant in vitro protective effect and to contribute to the strawberry tree honey antioxidant activity.

Protective effect of simple phenols from extravirgin olive oil against lipid peroxidation in intestinal Caco-2 cells.

Complex polyphenols present in extravirgin olive oil are not directly absorbed, but undergo gastrointestinal biotransformation, increasing the relative amount of tyrosol (TYR) and hydroxytyrosol (HT) entering the small and large intestine. We investigated the capacity of TYR and HT to inhibit the insult of dietary lipid hydroperoxydes on the intestinal mucosa, using cultures of Caco-2, a cell line with enterocyte-like features, and studying the effect of tert-butyl hydroperoxide (TBH) treatment on specific cell membrane lipid targets. The effect of homovanillic alcohol (HVA), metabolite of HT in humans and detected as metabolite of HT in Caco-2 cells, was also evaluated. Exposure to TBH induced a significant increase of the level of MDA, the formation of fatty acid hydroperoxides and 7-ketocholesterol and the loss of α-tocopherol. Pretreatment with both HT and HVA protected Caco-2 cells from oxidative damage: there was no significant detection of oxidation products and the level of α-tocopherol was preserved. Noteworthy, TYR also exerted a protective action against fatty acids degradation. In vitro trials, where the simple phenols were tested during linoleic acid and cholesterol oxidation, gave evidence of a direct scavenging of peroxyl radicals and suggested a hydrogen atom-donating activity.

Protective effect of hydroxytyrosol and its metabolite homovanillic alcohol on H(2)O(2) induced lipid peroxidation in renal tubular epithelial cells.

We investigated the capacity of hydroxytyrosol (HT), 3,4-dihydroxyphenylethanol, and homovanillic alcohol (HVA), 4-hydroxy-3-methoxy-phenylethanol, to inhibit H(2)O(2) induced oxidative damage in LLC-PK1, a porcine kidney epithelial cell line, studying the effect of H(2)O(2) on specific cell membrane lipid targets, unsaturated fatty acids and cholesterol. Exposure to H(2)O(2) induced a significant increase of the level of MDA together with a disruption of the membrane structure, with the loss of unsaturated fatty acids, cholesterol and alpha-tocopherol, and the formation of fatty acids hydroperoxides and 7-ketocholesterol. Pretreatment with HT protected renal cells from oxidative damage: the level of membrane lipids was preserved and there was no significant detection of oxidation products. HVA exerted a comparable activity, thus both HT and HVA were able to prevent in renal cells the lipid peroxidation process that plays a central role in tubular cell injury.

Protective effect of olive oil minor polar components against oxidative damage in rats treated with ferric-nitrilotriacetate.

The phenolic fraction of virgin olive oil exerts preventive effects against reactive oxygen species mediated degenerative diseases. To investigate its action as inhibitor of lipid peroxidation in vivo, we treated Wistar rats with olive oil minor polar components (MPC) (25-50 mg/kg bw) prior to the administration of a sublethal dose (15 mg Fe/kg bw) of ferric-nitrilotriacetate (Fe-NTA). Intraperitoneal injection (i.p.) of Fe-NTA lead to increased oxidative stress associated with extensive peroxidation of membrane lipids in plasma, kidney, and liver of treated rats. Fe-NTA treatment induced a significant decrease of the major oxidizable membrane lipids, alpha-tocopherol, fatty acids and cholesterol, together with an increase of fatty acids hydroperoxides (HP) and 7-ketocholesterol (7-keto). I.p. administration of MPC significantly inhibited fatty acids and cholesterol oxidation, and reduced the levels of HP and 7-keto. In summary, MPC administration protects organs against lipid peroxidation and conserves the endogenous alpha-tocopherol.

Cholesterol as target of Fe-NTA-induced lipid peroxidation in rat tissues.

Intraperitoneal injection of the iron-chelate, ferric-nitrilotriacetate (Fe-NTA), induces renal proximal tubular damage associated with oxidative damage in vivo. A sub-lethal dose of Fe-NTA (15 mg Fe/kg body weight) was administered IP to rats; animals were sacrificed and liver, kidney and plasma were collected 1-4 h after injection. In response to the Fe-NTA administration, there were significant time-dependent reductions of the levels of total lipids, cholesterol and total unsaturated fatty acids, and a rise in the concentrations of conjugated dienes, 7-ketocholesterol and fatty acids hydroperoxides, showing a pattern inversely correlated in plasma, kidney and liver. Cholesterol level decreased significantly from 1 h after injection in the kidney and 3-4 h in the plasma and liver of treated rats. This is the first report on cholesterol reduction and accumulated 7-ketocholesterol in the tissues of rats treated with Fe-NTA as a consequence of lipid peroxidation.