1H NMR metabolite fingerprint and pattern recognition of mullet (Mugil cephalus) bottarga

Nuclear magnetic resonance (NMR) spectroscopy combined with multivariate data analysis (MVA) was used to investigate the molecular components of the aqueous extract of samples of bottarga, that is, salted and dried mullet (Mugil cephalus) roe, manufactured in Sardinia (Italy) from mullets of known and unknown geographical provenience. Principal component analysis (PCA) applied to the processed (1)H NMR spectra indicated that samples tend to cluster according to their geographical origin and also on the basis of storage and manufacturing procedures. The most important metabolites that characterized grouping of samples are the free amino acids methionine (Met), glutamate (Glu), histidine (His), phenylalanine (Phe), tyrosine (Tyr), and isoleucine (Ile); trimethylamine (TMA) and dimethylamine (DMA), both biomarkers of degradation; nucleotides and derivatives; choline (Cho) and phosphorylcholine (P-cho); and lactate (Lac).     

Chemical composition of Greek avgotaracho prepared from mullet (Mugil cephalus): nutritional and health benefits

Crude composition, lipid composition, and tocopherols, ascorbic acid, cholesterol, phytosterols, and squalene content together with fatty acids and antiplatelet activities of total, neutral, and polar lipids of avgotaracho (wax-covered, dried, and salted Mugil cephalus roe) were studied and compared with those of similar products. Wax and steryl esters accounted for 63.7% of roe lipids followed by phosphatidylcholine (PC), which comprised 20.3%. Wax esters were rich in saturated fatty alcohols, monounsaturated fatty acids, and long chain omega3 highly unsaturated fatty acids (HUFA). The fatty acid distribution in roe total and neutral lipids was similar to that of wax esters, while in polar lipids, the omega3 HUFA predominated. Avgotaracho provides significant amounts of protein, fat, alpha-tocopherol, ascorbic acid, and PC, certain amounts of squalene and phytosterols, and cholesterol at levels comparable to hens' eggs. Total, polar, and neutral lipids of avgotaracho exhibited a strong inhibition of platelet activating factors and thrombin, with polar lipids being more active. The results obtained indicate that avgotaracho is a food of high nutritive value, rich in protein and lipids with a healthy lipid profile in terms of omega3/omega6 ratio and major fatty acid classes, while the antiplatelet activity of its oil indicates a putative antithrombotic potential.     

Characterization of the key aroma compounds in cooked grey mullet (Mugil cephalus) by application of aroma extract dilution analysis

Aroma and aroma-active compounds of wild grey mullet ( Mugil cephalus ) were analyzed by gas chromatography-mass spectrometry-olfactometry (GC-MS-O). According to sensory analysis, the aromatic extract obtained by simultaneous distillation and extraction (SDE) was representative of grey mullet odor. A total of 50 aroma compounds were identified and quantified in grey mullet. Aldehydes were qualitatively and quantitatively the most dominant volatiles in grey mullet. Aroma extract dilution analysis (AEDA) was used for the determination of aroma-active compounds of fish sample. A total of 29 aroma-active compounds were detected in aromatic extract of grey mullet, of which 24 were identified. On the basis of the flavor dilution (FD) factor, the most powerful aroma active compounds identified in the extract were (Z)-4-heptenal and nonanal, which were described as the strong cooked fish and green-fruity odor, respectively.     

Transport of Val-Leu-Pro-Val-Pro in human intestinal epithelial (Caco-2) cell monolayers

Angiotensin converting enzyme (ACE) inhibitory peptides are biologically active peptides that play a very important role in blood pressure regulation. In previous experiments, we obtained an ACE inhibitory peptide Val-Leu-Pro-Val-Pro (VLPVP) by DNA recombinant technology. The purpose of this study was to examine the bidirectional transport of VLPVP by using the human intestinal Caco-2 monolayers. The permeability coefficient ( P app) values of VLPVP over 4-8 mmol/L ranged from 7.44 x 10(-8) to 1.35 x 10(-6) cm/s for apical (AP) to basolateral (BL) transport, while the P app values for BL to AP flux were significantly lower than those for the AP to BL flux, with efflux ratio values of 0.74-0.13 over 4-8 mM. Preincubation of the paracellular transport enhancer (sodium deoxycholate), the inhibitor of multidrug resistant protein (MK-571), or sodium azide stimulated efflux of VLPVP significantly ( p < 0.01); these results indicate that the transport of VLPVP across Caco-2 monolayers was involved in paracellular diffusion and MRP2 transport.     

Differential effects of flavonoids on barrier integrity in human intestinal Caco-2 cells

Flavonoids, present in fruits, vegetables, and teas, provide beneficial effects for our health. We investigated the effect of a number of flavonoids on tight junction (TJ) barrier integrity in human intestinal Caco-2 cells. Transepithelial electrical resistance (TER; a TJ integrity marker) across cell monolayers was measured in cells incubated with flavonoids for 24 h. Chrysin decreased the TER, indicating a decrease in TJ integrity. Daidzein, hesperetin, naringenin, and morin increased the TER, indicating increased TJ integrity. Luteolin and genistein increased or normalized the TER after a transient decrease. Immunoblot analysis revealed that these changes in TER were caused by modification of the cytoskeletal association and expression of TJ proteins, zonula occludens (ZO)-1, ZO-2, occludin, junctional adhesion molecule-1, and/or claudins. Our results suggest that various flavonoids participate in the regulation of intestinal TJ barrier integrity and that this regulation may partially contribute to the flavonoid-mediated biological effects on our health.     

Grape seed extract affects proliferation and differentiation of human intestinal Caco-2 cells

The effect of daily contact of a grape seed extract (GSE) on Caco-2 cell proliferation and differentiation was investigated. GSE at 400 mg/L was added to Caco-2 cells for 2 h a day after successive incubation in saliva, gastric, and pancreatic media. When applied at the beginning of the cell culture, GSE triggered inhibition of cell growth associated with a possible cytotoxic reaction. On the other hand, when the treatment was applied to confluent cells, treated cells displayed a higher protein content than control cells and a more developed brush border, with taller and denser microvilli. These observations were accompanied by stimulation of alkaline phosphatase activity, especially at day 5 postconfluency, with a 2.2-fold increase in comparison with the control. On the other hand, aminopeptidase N activity was inhibited throughout the differentiation period in GSE-treated cells to reach 28.8% of control cell activity on day 30. GSE did not affect either sucrase-isomaltase activity or cytoplasmic lactate dehydrogenase (LDH) activity, which otherwise appeared to be a good cellular marker. GSE treatment of Caco-2 cells thus inhibited their proliferation from seeding onward and stimulated both proliferation and differentiation after confluency.     

Effect of bioactive dietary polyphenols on zinc transport across the intestinal Caco-2 cell monolayers

Polyphenolic compounds are known to possess many beneficial health effects, including the antioxidative activities of scavenging reactive oxygen species and chelating metals, such as iron and zinc. Tea and red wine are thought to be important sources of these compounds. However, some polyphenolic compounds can also reduce the absorption of iron, and possibly other trace metals, when included in a diet. There is very little information on the effect of dietary polyphenolic compounds on the status of trace elements other than iron. The effects of epigallocatechin-3-gallate (EGCG), green tea extract (GT), and grape seed extract (GSE) on the absorption of (65)Zn were examined and compared with their effects on (55)Fe absorption in human intestinal Caco-2 cells grown on microporous membrane inserts. The levels of EGCG, GT, and GSE used in this study were within physiological ranges and did not affect the integrity of the Caco-2 cell monolayers. GSE significantly (P < 0.05) reduced zinc transport across the cell monolayer, and the decreased zinc transport was associated with a reduction in apical zinc uptake. However, EGCG and GT did not alter zinc absorption. In contrast, the polyphenolic compounds in EGCG, GT, and GSE almost completely blocked transepithelial iron transport across the cell monolayer. The effect of GSE on zinc absorption was very different from that on iron absorption. Whereas GSE decreased zinc absorption by reducing apical zinc uptake, the polyphenolic compounds inhibited iron absorption by enhancing apical iron uptake. GSE inhibited zinc absorption similarly to that observed for phytate. Phytate significantly (P < 0.05) decreased transepithelial zinc transport by reducing apical zinc uptake. The inhibition of zinc absorption may be due to the presence of procyanidins in GSE, which bind zinc with high affinity and block the transport of zinc across the apical membrane of enterocytes. Further research on the absorption of zinc as zinc-polyphenol complexes and free zinc should provide further insight into the process of dietary zinc absorption in the presence of GSE and other bioactive dietary polyphenols. The present study suggests that some individuals should consider their zinc status if they regularly consume procyanidin-containing foods in their diet. However, further studies, especially in vivo studies, are needed to confirm these results.     

Cellular uptake and efflux of trans-piceid and its aglycone trans-resveratrol on the apical membrane of human intestinal Caco-2 cells

Two stilbenes (trans-piceid and its aglycone trans-resveratrol) were investigated in the uptake across the apical membrane of the human intestinal cell line Caco-2 in order to determine their mechanisms of transport. The uptake was quantified using a reverse phase high-performance liquid chromatography method with fluorescence detection. The rate of cellular accumulation in the cells was found to be higher for trans-resveratrol than for trans-piceid. In addition, trans-resveratrol uses passive transport to cross the apical membrane of the cells, whereas the transport of trans-piceid is likely active. With regard to the mechanisms of transport, the involvement of the active transporter SGLT1 in the absorption of trans-piceid was deduced using various inhibitors directly or indirectly exploiting the activity of this transporter (glucose, phlorizin, and ouabain). Moreover, we investigated the involvement of the multidrug-related protein 2 (MRP2), an efflux pump present on the apical membrane, in stilbene efflux by Caco-2 cells. The effect of MK-571 (an MRP inhibitor) seems to implicate MRP2 as responsible for apical efflux of trans-piceid and trans-resveratrol.     

Variation of chemical composition of the lipophilic extracts from yellow birch (Betula alleghaniensis) foliage

The occurrence of biologically active compounds identified for the first time in the lipophilic extracts of yellow birch (Betula alleghaniensis Britt.) foliage led to the quantification of the seasonal variation of their concentrations. Yellow birch foliage was collected from late June until late September 2003 in two different regions of Quebec. The extraction yields using hexane as a solvent were determined, and the extracts were analyzed by GC-MS to identify their molecular composition. In terms of both extraction yields and the concentration of the targeted molecules present in the extracts, mid-September has been determined as the best time to collect foliage samples. A total of 14 constituents were identified in these extracts. This is the first report of the presence of all of these constituents in yellow birch foliage and of some of them in the genus Betula. The most important compounds identified in yellow birch foliage extracts are triterpene squalene and aliphatic hydrocarbon tetracosan, aliphatic alcohol phytol, fatty acids hexadecanoic and octadecanoic, pentacyclic triterpenes alpha- and beta-amyrin, and phytosterol stigmast-5-en-3-ol.     

Effect of gamma-irradiation on the lipid profile of nutmeg (Myristica fragrans Houtt.)

The effect of gamma-irradiation on the lipid constituents of nutmeg (Myristica fragrans) was examined at radiation doses between 2.5 and 10 kGy. The fatty acid composition of the triacylglycerol, the major lipid component, was found to be made up of myristic (90%), palmitic (6%), lauric (3%), petroselinic (0.13%), and stearic acids (0.5%) as determined by gas chromatography-mass spectrometry. A dose-dependent decrease in the triacylglycerol content and a concomitant increase in free fatty acids characterized the lipid profile of the irradiated spice. This suggested a breakdown of acylglycerols during radiation processing, resulting in the release of free fatty acids. These changes were found to be significant at doses above 5 kGy. The impact of the above changes on the flavor of the spice is discussed. These studies suggest that radiation processing of nutmeg should be limited to a dose of 5 kGy.     

Digestive Stability, micellarization, and uptake of beta-carotene isomers by Caco-2 human intestinal cells

While isomeric profiles of carotenoids found in food often differ from those in body fluids and tissues, insights about the basis for these differences remain limited. We investigated the digestive stability, relative efficiency of micellarization, and cellular accumulation of trans and cis isomers of beta-carotene (BC) using an in vitro digestion procedure coupled with human intestinal (Caco-2) cells. A meal containing applesauce, corn oil, and either water-soluble beadlets (WSB) or Dunaliella salina (DS) as a BC source was subjected to simulated gastric and small intestinal digestion. BC isomers were stable during digestion, and the efficiency of micellarization of cis-BC isomers exceeded that of all-trans-BC isomers. The cellular profile of carotenoids generally reflected that in micelles generated during digestion, and intracellular isomerization was minimal. These data suggest that cis isomers of BC are preferentially micellarized during digestion and transferred across the brush-border surface of the enterocyte from mixed micelles with similar efficiency as all-trans-BC at the concentrations of the carotenoids utilized in this study.     

Potential of rosemary (Rosemarinus officinalis L.) diterpenes in preventing lipid hydroperoxide-mediated oxidative stress in Caco-2 cells

The effects of 24 h supplementation of Caco-2 cells with carnosic acid and carnosol, and their activities against 5 microM oleic acid hydroperoxide (OAHPx)-mediated oxidative stress, were investigated. At 24 h of incubation, under nonstressed and stressed conditions, both compounds at 25, 50, and 100 microM supplement concentrations reduced catalase activity, whereas changes in glutathione peroxidase and superoxide dismutase activities varied depending upon the concentrations. Relative to control cultures, carnosic acid and carnosol reduced membrane damage by 40-50% when stressed by OAHPx. Carnosic acid and carnosol inhibited lipid peroxidation by 88-100% and 38-89%, respectively, under oxidative stress conditions. Both compounds significantly lowered DNA damage induced by OAHPx. Results of this study suggest that antioxidant activities of carnosic acid and carnosol could be partly due to their ability to increase or maintain glutathione peroxidase and superoxide dismutase activities.     

Effect of food models and low-temperature storage on the adhesion of Lactobacillus rhamnosus GG to Caco-2 cells

This study evaluated the effects of fat and sugar levels on the surface properties of Lactobacillus rhamnosus GG during storage in food model systems, simulating yogurt and ice cream, and related them with the ability of the bacterial cells to adhere to Caco-2 cells. Freeze-dried L. rhamnosus GG cells were added to the model food systems and stored for 7 days. The bacterial cells were analyzed for cell viability, hydrophobicity, ζ potential, and their ability to adhere to Caco-2 cells. The results indicated that the food type and its composition affected the surface and adhesion properties of the bacterial cells during storage, with yogurt being a better delivery vehicle than ice cream in terms of bacterial adhesion to Caco-2 cells. The most important factor influencing bacterial adhesion was the storage time rather than the levels of fats and sugars, indicating that conformational changes were taking place on the surface of the bacterial cells during storage.     

Effect of centrifugal ultrafiltration on the composition of aqueous extracts of saffron spice (Crocus sativus L.)

The purpose of this research was to study the effect of centrifugal ultrafiltration (UF) on the composition of aqueous extracts of saffron spice. The contents of seven crocetin esters, picrocrocin, and two kaempferol glycosides were analyzed by UV-vis and HPLC in the filtrate and retentate fractions from 16 centrifugal filter devices with regenerated cellulose (RC) and polyethersulfone (PES) membranes ranging from 1-100 kDa nominal molecular weight cutoff (MWCO). The separation of crocetin esters from picrocrocin and their concentration with centrifugal UF have been demonstrated. A great heterogeneity of results regarding devices with equal MWCO was found that could not be related to the membrane material or manufacturer. Four devices of 5 and 10 kDa MWCO, three of which had RC membranes, showed the best results. The device having the lowest MWCO also showed a potential to obtain picrocrocin without crocetin esters and could be considered in successive UF steps. The less polar crocetin esters were rejected better than the others.     

Microbial metabolites of ingested caffeic acid are absorbed by the monocarboxylic acid transporter (MCT) in intestinal Caco-2 cell monolayers

It was previously reported that m-coumaric acid, m-hydroxyphenylpropionic acid (mHPP), and 3,4-dihydroxyphenylpropionic acid (DHPP) are major metabolites of ingested caffeic acid formed by gut microflora and would be transported by the monocarboxylic acid transporter (MCT). We have directly measured their absorption characteristics in Caco-2 cells using a coulometric detection method involving HPLC-ECD. The proton-coupled directional transport of m-coumaric acid, mHPP, and DHPP was observed, and the transport was inhibited by an MCT substrate. The permeation of m-coumaric acid and mHPP was concentration-dependent and saturable: The Michaelis constant for m-coumaric acid and mHPP was 32.5 and 12.9 mM, respectively, and the maximum velocity for m-coumaric acid and mHPP was 204.3 and 91.2 nmol (min)(-1) (mg protein)(-1), respectively. By contrast, the permeation of DHPP was nonsaturable even at 30 mM and was inversely correlated with the paracellular permeability of Caco-2 cells. Our results demonstrate that these compounds are absorbed by the MCT, although DHPP is mainly permeated across Caco-2 cells via the paracellular pathway. MCT-mediated absorption of phenolic compounds per se and their colonic metabolites would exert significant impact on human health.     

Lactic acid decreases Fe(II) and Fe(III) retention but increases Fe(III) transepithelial transfer by Caco-2 cells

Lactic acid (LA) has been proposed to be an enhancer for dietary iron absorption, but contradictory results have also been reported. In the present study, fully differentiated Caco-2 cell monolayers were used to evaluate the effects of LA (1-50 mmol/L) on the cellular retention and transepithelial transport of soluble non-heme iron (as ferric nitrilotriacetate). Our data revealed a linear decline in Fe(III) retention with respect to the concentration of LA added. In the presence of 50 mmol/L LA, retention of Fe(III) and Fe(II) decreased 57% and 58%, respectively. In contrast, transfer of Fe(III) across the cell monolayer was doubled, while Fe(II) transfer across the cell monolayer decreased 35%. We conclude that LA reduces cellular retention and transepithelial transport of Fe(II) by Caco-2 cells in a dose-dependent manner. However, while LA also reduces retention of Fe(III) by Caco-2 cells, the transfer of Fe(III) across cell monolayers is enhanced, possibly due to effects on paracellular transport.     

Bioaccessibility of phenols in common beans ( Phaseolus vulgaris L.) and iron (Fe) availability to Caco-2 cells

Samples of common and biofortified beans ( Phaseolus vulgaris ), both raw and cooked (autoclaved at 120 degrees C for 20 min) were analyzed for their polyphenol composition. Polyphenols were identified via HPLC-UV/diode array detection. Cooking favored the extraction of polyphenols without the need of a hydrolysis step, a fact that is of interest because this is the usual form in which beans are consumed. The main differences between white and colored beans were the presence of free kaempferol (13.5-29.9 microg g(-1)) and derivatives (kaempferol-3-O-glucoside) (12.5-167.5 microg g(-1)), only in red and black beans. An in vitro digestion (pepsin, pH2; pancreatin-bile extract, pH 7) was applied to beans to estimate bioaccessibility of individual polyphenols. Kaempferol from seed coats exhibited high bioaccessibility (45.4-62.1%) and a potent inhibitor effect on Fe uptake at concentrations ranging from 0.37 to 1.30 microM. Caco-2 cell ferritin formation was used to evaluate Fe uptake. Cell Fe uptake was significant only from white beans.     

Transport of cranberry A-type procyanidin dimers, trimers, and tetramers across monolayers of human intestinal epithelial Caco-2 cells

A-type procyanidin oligomers in cranberries are known to inhibit the adhesion of uropathogenic bacteria. B-type procyanidin dimers and trimers are absorbed by humans. The absorption of A-type procyanidins from cranberries in humans has not been demonstrated. This study examined the transport of A-type cranberry procyanidin dimers, trimers, and tetramers on differentiated human intestinal epithelial Caco-2 cell monolayers. Procyanidins were extracted from cranberries and purified using chromatographic methods. Fraction I contained predominantly A-type procyanidin dimer A2 [epicatechin-(2-O-7, 4-8)-epicatechin]. Fraction II contained primarily A-type trimers and tetramers, with B-type trimers, A-type pentamers, and A-type hexamers being minor components. Fraction I or II in solution was added onto the apical side of the Caco-2 cell membranes. The media at the basolateral side of the membranes were analyzed using HPLC-MS(n) after 2 h. Data indicated that procyanidin dimer A2 in fraction I and A-type trimers and tetramers in fraction II traversed across Caco-2 cell monolayers with transport ratio of 0.6%, 0.4%, and 0.2%, respectively. This study demonstrated that A-type dimers, trimers, and tetramers were transported across Caco-2 cells at low rates, suggesting that they could be absorbed by humans after cranberry consumption.     

Effects of diosmin, a flavonoid glycoside in citrus fruits, on P-glycoprotein-mediated drug efflux in human intestinal Caco-2 cells

The effects of citrus flavonoids on P-glycoprotein (P-gp)-mediated drug efflux were examined in human intestinal Caco-2 cells. The cellular accumulation of rhodamine-123 was measured using 10 citrus flavonoids for preliminary screening. Among the flavonoids tested, diosmin significantly increased the accumulation of rhodamine-123 in Caco-2 cells. In the bidirectional transport of digoxin, diosmin increased the apical-to-basal (A-to-B) transport but decreased the basal-to-apical (B-to-A) transport in both concentration- and time-dependent manners. The digoxin transport ratio (B-A/A-B) was estimated to be 2.3 at a concentration of 50 microM of diosmin, which was significantly lower than the 15.2 found in the control. The apparent Ki values for P(app,A-B) and P(app,B-A) were 16.1 and 5.7 microM, respectively. These results demonstrated that diosmin effectively inhibited the P-gp-mediated efflux in Caco-2 cells. Diosmin is one of the main components in citrus fruits, and the intake of food supplements containing this compound may potentially increase the absorption of drugs able to act as P-gp substrates. The clinical relevance of this interaction should be further evaluated using in vivo experiments.     

Tea polyphenols inhibit the transport of dietary phenolic acids mediated by the monocarboxylic acid transporter (MCT) in intestinal Caco-2 cell monolayers

It was previously reported that a fluorescent marker dye, fluorescein, is transported via the monocarboxylic acid transporter (MCT). Fluorescein transport was competitively inhibited by MCT substrates such as ferulic and salicylic acids. Tea polyphenols, in particular, epigallocatechin gallate (EGCg) and epicatechin gallate (ECg), inhibited the transport of fluorescein. Tea polyphenols also inhibited the transport of salicylic and ferulic acids, suggesting tea polyphenols might be substrates of MCT. However, the transepithelial flux of tea polyphenols was much lower than that of the MCT substrates and was inversely correlated with the paracellular permeability of Caco-2 cell monolayers. These findings suggest that tea polyphenols are not substrates but inhibitors of MCT. Furthermore, the transepithelial transport of these polyphenols is mainly via paracellular diffusion. However, directional transport of ECg and EGCg from the basolateral to the apical side was observed, indicating that the behavior of tea polyphenols in the intestinal epithelium is complex.