Quantitation of flavonoid constituents in citrus fruits.

Twenty-four flavonoids have been determined in 66 Citrus species and near-citrus relatives, grown in the same field and year, by means of reversed phase high-performance liquid chromatography analysis. Statistical methods have been applied to find relations among the species. The F ratios of 21 flavonoids obtained by applying ANOVA analysis are significant, indicating that a classification of the species using these variables is reasonable to pursue. Principal component analysis revealed that the distributions of Citrus species belonging to different classes were largely in accordance with Tanaka's classification system.     

Polymethoxylated flavones and other phenolic derivates from citrus in their inhibitory effects on P-glycoprotein-mediated transport of talinolol in Caco-2 cells

Many studies investigating drug interactions with citrus compounds focus on the major grapefruit furanocoumarins bergamottin, dihydroxybergamottin, and the flavonoid naringenin. This study evaluated the influence of polymethoxylated flavones (PMFs), tangeretin, nobiletin, 3,5,6,7,8,3,4'-heptamethoxyflavone, and sinensetin, as well as other minor occurring citrus phenols, hesperetin, limettin, 7-OH-coumarin, 7-geranyloxycoumarin, and eriodictyol, on P-glycoprotein-mediated transport of the beta-blocker talinolol using the Caco-2 cell monolayer model and was used to determine the structure-function aspects of the interaction. The transport of talinolol across Caco-2 cells monolayers was determined in the absence and presence of distinct concentrations of the calcium-channel blocker verapamil (a known inhibitor of P-glycoprotein) and citrus compounds. A sigmoid dose-response model was used to fit the data and to estimate the IC50 values of the potential inhibitors. Results from this study show that PMFs significantly decreased talinolol transport from the basolateral to apical side, where tangeretin had the lowest IC50 of 3.2 micromol/L, followed by nobiletin, heptamethoxyflavone, and sinensetin with IC50 values of 3.5, 3.8, and 3.9 micromol/L, respectively. However, the efficacy of the compounds did not appear to be dependent on the number of methoxy groups. Other citrus compounds did not have any significant effect on the transport of talinolol. This study suggests that PMFs have a high potential in the interaction with P-gp-mediated talinolol transport in Caco-2 cells. Based on their relatively low concentrations (< or =3 microg/mL) in citrus, the clinical relevance of these interactions needs to be further elucidated in in vivo studies.     

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.     

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.     

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.     

Chicken eggshell matrix proteins enhance calcium transport in the human intestinal epithelial cells, Caco-2

Chicken eggshell powder has been proposed as an attractive source of calcium for human health to increase bone mineral density in an elderly population with osteoporosis. However, factors affecting calcium transport of eggshell calcium have not yet been evaluated. Chicken eggshell contains about 1.0% (w/w) matrix proteins in addition to a major form of calcium carbonate (95%, w/w). In this study, we found that soluble eggshell matrix proteins remarkably enhance calcium transport using in vitro Caco-2 cell monolayers grown on a permeable support. The total calcium transport across Caco-2 monolayers showed an increase of 64% in the presence of 100 microg/well soluble eggshell matrix proteins. The active enhancer with a molecular mass of 21 kDa was isolated by reversed phase high-performance liquid chromatography and did not correspond to any previously identified protein. The N-terminal sequence was determined to be Met-Ala-Val-Pro-Gln-Thr-Met-Val-Gln. The possible mechanisms of eggshell matrix protein-mediated increase in calcium transport and the potential significance of eggshell calcium as a nutraceutical are discussed.     

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.     

Sodium copper chlorophyllin: in vitro digestive stability and accumulation by Caco-2 human intestinal cells

Sodium copper chlorophyllin (SCC), a mixture of water-soluble chlorophyll derivatives, is used as both a food colorant and a common dietary supplement. Although the potential antimutagenic and antioxidant properties of this commercial preparation have been demonstrated, limited information is available on its digestion and absorption by humans. Stability of SCC was examined during simulated gastric and small intestinal digestion. Three preparations were subjected to in vitro digestion: SCC in water, SCC in water + 10% corn oil, and SCC in applesauce. SCC components from raw material preparations and in digested samples were analyzed by C(18) HPLC with photodiode array detection. Cu(II)chlorin e(4), the major chlorin component of SCC, was relatively stable during simulated digestion. In contrast, greater than 90% of Cu(II)chlorin e(6) was degraded to undetermined products during digestion. Recovery of Cu(II)chlorin e(6) after digestion was increased by incorporation of SCC into applesauce, suggesting a protective role of the inclusion matrix for stabilization of labile SCC components. Accumulation of SCC derivatives was investigated by using differentiated cultures of the TC7 clone of the Caco-2 human intestinal cell line. Cellular accumulation from media containing 0.5 to 60 ppm SCC was linear with intracellular content ranging between 0.2 and 29.6 microg of total SCC per mg of cellular protein. Uptake of SCC by Caco-2 cells was significantly (p < 0.01) lower in cultures incubated at 4 degrees C than in those incubated at 37 degrees C. Although intracellular SCC was transported into both apical and basolateral compartments when Caco-2 cells were grown on inserts, apical efflux was significantly greater (p < 0.01) than basolateral efflux. Stability of Cu(II)chlorin e(4) during in vitro digestion and effective uptake by Caco-2 enterocyte-like cells support the likelihood that a portion of this SCC component or its metabolites is absorbed from the human intestine.     

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.     

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.     

Transepithelial transport of alpha-lipoic acid across human intestinal Caco-2 cell monolayers

Alpha-lipoic acid (LA) is used in dietary supplements or food with antioxidative functions. The mechanism for the intestinal absorption of alpha-lipoic acid was investigated in this study by using human intestinal Caco-2 cell monolayers. LA was rapidly transported across the Caco-2 cell monolayers, this transport being energy-dependent, suggesting transporter-mediated transport to be the mechanism involved. The LA transport was strongly dependent on the pH value, being accelerated in the acidic pH range. Furthermore, such monocarboxylic acids as benzoic acid and medium-chain fatty acids significantly inhibited LA transport, suggesting that a proton-linked monocarboxylic acid transporter (MCT) was involved in the intestinal transport of LA. The conversion of LA to the more antioxidative dihydrolipoic acid was also apparent during the transport process.     

Mass spectrometric identification and high-performance liquid chromatographic determination of a flavonoid glycoside naringin in human urine

The aim of this study was to evaluate the absorption of a citrus flavonoid, naringin, as its glycosylated form. Six healthy volunteers (three males and three females) were studied. After a single oral administeration of 500 mg of naringin, intact naringin was isolated from 2-4 h urine. Isolated naringin was identified by the LC/electrospray ionization mass spectrometry (ESI-MS), MS/MS, and MS/MS/MS techniques. The cumulative urinary excretion of naringin and its metabolites (naringenin and naringenin glucuronides) was determined by HPLC for 0-24 h. Approximately 0.02% of the administered dose was recovered in urine as unchanged naringin, whereas urinary recoveries of naringenin and naringenin glucuronides were approximately 0.4 and 3.6% of the administered dose, respectively. It was concluded that trace amounts of orally administered naringin can be absorbed as the glycoside. However, it is not clear whether the glycoside is cleaved before or after absorption to generate naringenin.     

Absorption of black currant anthocyanins by monolayers of human intestinal epithelial Caco-2 cells mounted in ussing type chambers

Anthocyanins (ACNs) have been reported to have multiple biological properties imparting benefits to human health. Their role in human nutrition, however, needs to be related to biokinetic data, such as bioavailability. The purpose of the present study was to focus on the potential absorption of black currant ( Ribes nigrum L.) ACNs. Caco-2 monolayers were used as an in vitro model of the absorptive intestinal epithelium. For absorption studies, Caco-2 cells grown on permeable filters were mounted into Ussing type chambers. The monolayer integrity was monitored by measuring the transepithelial electrical resistance (TEER). Luminal to serosal transport of ACNs was examined by comparing ACN disappearance from the luminal solution of Ussing chambers not containing any inserts (control chambers) with that of Ussing chambers containing inserts. ACNs (C total ACN approximately 180 microM) were not detected in any serosal solution. However, it was shown that ACNs disappeared from the luminal side, not due to ACN degradation processes but rather--at least in part--due to physiological actions of the cells. The luminal net disappearance of ACNs was calculated (max(t20 min) approximately 11% for total ACNs) and labeled as "absorption efficiency". This apical transport might occur to a much larger extent than the further translocation across the basolateral membrane. Thus, cell metabolism and translocation across the basolateral membrane may be the key determinants of ACN absorption and bioavailability.     

Uptake of 2S albumin allergens, Ber e 1 and Ses i 1, across human intestinal epithelial Caco-2 cell monolayers

We have investigated the absorption rates of two purified major allergen 2S albumins, Ber e 1 from Brazil nuts (Bertholletia excelsa Humb. & Bonpl.) and Ses i 1 from white sesame seeds (Sesamum indicum L.), across human intestinal epithelial Caco-2 cell monolayers following gastrointestinal digestion in vitro. The transport from apical to basolateral side in cell monolayers was evaluated by RP-HPLC-UV and indirect competitive ELISA methods, being confirmed by western-blotting analysis. Significant amounts (approximately 15-25 nmol micromol(-1) initial amount/h) of intact Ber e 1 and Ses i 1 were found in the basolateral side. The absorption rates of both plant allergens through the cell monolayer were shown to be constant during the whole incubation period (4 h at 37 degrees C), verifying that the permeability of the membrane was not altered by the allergen digests. Our findings revealed that both purified 2S albumin allergens may be able to survive in immunologically reactive forms to the simulated harsh conditions of the gastrointestinal tract to be transported across the Caco-2 cell monolayers, so that they would be able to sensitize the mucosal immune system and/or elicit an allergic response.     

Determination of oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with tandem mass spectrometry

A new methodology is described for rapidly determining the herbicide oryzalin in water, citrus fruits, and stone fruits by liquid chromatography with negative ion electrospray ionization tandem mass spectrometry (LC/MS/MS). Oryzalin is extracted from water using a polymeric sorbent solid phase extraction (SPE) column and from fruit using methanol. The water samples require no further purification, but an aliquot of the fruit sample extracts is diluted with water and purified using a polymeric 96 well SPE plate. Purified extracts are concentrated prior to determination by LC/MS/MS at m/z 345 (Q1) and m/z 281 (Q3) using an external standard for calibration. The validated limits of quantitation were 0.05 microg/L in water (drinking water, surface water, and groundwater) and 0.01 microg/g in citrus fruits (oranges and lemons) and stone fruits (peaches and cherries). Recoveries averaged 102% for water samples and 85-89% for the various types of fruit samples. For all fortification levels combined, the relative standard deviations ranged from 4 to 6% for water and from 2 to 4% for fruit.     

Protein changes in the albedo of citrus fruits on postharvesting storage

In this work, major protein changes in the albedo of the fruit peel of Murcott tangor (tangerine x sweet orange) during postharvest ageing were studied through 2D PAGE. Protein content in matured on-tree fruits and in fruits stored in nonstressing [99% relative humidity (RH) and 25 degrees C], cold (99% RH and 4 degrees C), and drought (60% RH and 25 degrees C) conditions was initially determined. Protein identification through MS/MS determinations revealed in all samples analyzed the occurrence of manganese superoxide dismutase (Mn SOD), actin, ATP synthase beta subunit (ATPase), citrus salt-stress associated protein (CitSap), ascorbate peroxidase (APX), translationally controlled tumor protein (TCTP), and a cysteine proteinase (CP) of the papain family. The latter protein was identified in two different gel spots, with different molecular mass, suggesting the simultaneous presence of the proteinase precursor and its active form. While Mn SOD, actin, ATPase, and CitSap were unchanged in the assayed conditions, TCTP and APX were downregulated during the postharvest ageing process. Ageing-induced APX repression was also reversed by drought. CP contents in albedo, which were similar in on- and off-tree fruits, were strongly dependent upon cold storage. The active/total CP protein ratio significantly increased after cold exposure. This proteomic survey indicates that major changes in protein content in the albedo of the peel of postharvest stored citrus fruits are apparently related to the activation of programmed cell death (PCD).     

Ethanol and polyphenolic free wine matrix stimulate the differentiation of human intestinal Caco-2 cells. Influence of their association with a procyanidin-rich grape seed extract

The effect of daily contact with ethanol on Caco-2 cell differentiation was investigated. Pure ethanol (1%) and a polyphenolic free wine matrix (polyphenol-free wine containing 1% ethanol) associated or not with a procyanidin-rich grape seed extract (GSE) were added to Caco-2 cells from confluency for 2 h a day after successive incubation in salivary, gastric, and pancreatic media. Treatment with 1% ethanol did not appear to be cytotoxic to cells, but it also stimulated Caco-2 cell differentiation, particularly in the first days following confluency, and this effect was more marked when associated with polyphenolic free wine matrix constituents. This activation resulted in an increase in microvillar density, organization, and elongation (+70%) and was associated with strong stimulation of sucrase-isomaltase (+780%) and a concomitant regular increase in cell protein content (+50-88%). While the presence of GSE in alcoholic solutions did not modify the morphological pattern observed in cells subjected to ethanol and polyphenolic free wine matrix alone, it had a clear reducing effect on their microvillus elongation (-30%). However, these stimulating effects of ethanol on morphological differentiation were attenuated from day 10 postconfluency, which could suggest cell cytoprotection against ethanol. These are the first results in support of the notion that moderate concentration of ethanol may stimulate the differentiation of Caco-2 cells, particularly when integrated with a polyphenolic free wine matrix.     

Effect of variety, processing, and storage on the flavonoid glycoside content and composition of lettuce and endive

Eight varieties of lettuce (Lactuca sativum) and three varieties of endive (Cichorium endivia) were analyzed for flavonoid composition and content. Total flavonoid contents, expressed as units of aglycon for fresh material, were in the ranges of 0.3-229 microg/g for lettuce and 44-248 microg/g for endive. Five quercetin conjugates [quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin 3-O-glucuronide, quercetin 3-O-(6-O-malonyl)glucoside, and quercetin 3-O-rhamnoside] and luteolin 7-O-glucuronide were measured in the green-leafed lettuce and an additional two cyanidin conjugates [cyanidin 3-O-glucoside and cyanidin 3-O-[(6-O-malonyl)glucoside]] in the red-leafed varieties. Three kaempferol conjugates [kaempferol 3-O-glucoside, kaempferol 3-O-glucuronide, and kaempferol 3-O-[6-O-malonyl)glucoside]] were measured in each of the endive varieties. The presence and identity of kaempferol 3-O-(6-O-malonyl)glucoside in endive was shown for the first time. Shredding of lettuce leaf followed by exposure to light produced significant losses of the flavonoid moiety in the green oak leaf (94%), red oak leaf (43%), iceberg (36%), green batavia (25%), lollo biondo (24%), and lollo rosso (6%) samples, whereas cos and green salad bowl samples did not show an overall loss. Shredding of endive also produced loss of the flavonoid moiety in escarole (32%), fine frisee (13%), and coarse frisee (8%). Significant demalonation was observed for both the quercetin and cyanidin glucosides in lettuce, whereas a similar degradation of the kaempferol analogue was found in endive tissue. Storage of whole heads of both lettuce and endive in the dark at 1 degrees C and 98% humidity for 7 days resulted in losses of total flavonol glycosides in the range of 7-46%. The identification of the amounts, position of substitution, and nature of the sugars is important for understanding the potential bioavailability and biological activities of flavonoids in salads.     

Distribution of various peptides in citrus fruits (grapefruit, lemon, and orange)

Tissue- and species-specific peptides of the grapefruit have been investigated by SDS-PAGE and Western blot. Five peptides from the juice and one peptide from the peel were isolated by preparative gel electrophoresis. Polyclonal antibodies were developed against them in mice. It can be established that 82, 63, and 46 kDa peptides occurred exclusively in the samples prepared from the grapefruit and the lemon juice, whereas in the orange juice, only the 82 kDa peptide could be detected. The 31 kDa peptide is characteristic for the peel samples of grapefruit and lemon. The 210 kDa peptide did not show any specificity. A 117 kDa peptide appeared in the juice and peel of grapefruit and in the peel of lemon but not in the orange. From the data of this study, it is supposed that some of the polyclonal antibodies developed against characteristic juice and peel peptides can be used to test commercial grapefruit juice products for adulteration.     

Transepithelial transport of microbial metabolites of quercetin in intestinal Caco-2 cell monolayers

m-Hydroxyphenylacetic acid (mHPA), 3,4-dihydroxyphenylacetic acid (DHPA), and 4-hydroxy-3-methoxyphenylacetic acid (HMPA) are major microbial metabolites of quercetin. After administration of quercetin to human subjects, these metabolites are readily detected in blood and urine. mHPA, DHPA, and HMPA are thought to exert protective biological activity within the body due to their antioxidant properties. However, very little work has been published concerning their absorption. I have examined the absorption characteristics of the quercetin metabolites in Caco-2 cells by a coulometric detection method using HPLC-ECD. All of them exhibited nonsaturable transport in Caco-2 cells up to 30 mM, whereas HMPA and mHPA also showed proton-coupled polarized absorption. The proton-coupled directional transport of HMPA and mHPA was inhibited by the substrate of the monocarboxylic acid transporter (MCT). A considerable amount of apically loaded HMPA and mHPA was taken up and transported through to the basolateral side, while almost all of the apically loaded DHPA was retained on the apical side. Furthermore, the transepithelial flux of DHPA was inversely correlated with the paracellular permeability of Caco-2 cells, although those of HMPA and mHPA were almost constant. These results indicate that transport of DHPA was mainly via paracellular diffusion, although HMPA and mHPA were absorbed to some extent by the MCT.