Human Intestinal Caco-2 Cell Monolayers Research Articles

In Vitro Absorption of Dietary trans-Resveratrol from Boiled and Roasted Peanuts in Caco-2 Cells

Previous studies on the transport and absorption of resveratrol (3,5,4-O-trihydroxystilbene) were done using the pure compound. In this study, the absorption of resveratrol in digested peanut micellar from boiled and roasted peanuts was investigated using a human intestinal Caco-2 cell monolayer. The amount transported and rate of transport of both resveratrol glycosides and its hydrolytic product were quantified by a reverse-phase high-performance liquid chromatography method with mass spectrometric detection. Four peaks were identified in the digested peanut micellar of both boiled and roasted peanuts: two resveratrol glycosides, one resveratrol diglycoside, and possibly an acylated resveratrol glycoside. Resveratrol from roasted peanut micellar had a higher transport rate than those from the boiled peanut. This implies that resveratrol from roasted peanut is better absorbed than from boiled peanut. Also, the rate of transport and amount of resveratrol transported were higher for the hydrolytic product than the nonhydrolyzed glycosides. This has strong implications for in vivo absorption as the enzymatic activity of gut microflora could enhance the bioavailability of β-glycosides of dietary polyphenols.

Permeability of rosmarinic acid in Prunella vulgaris and ursolic acid in Salvia officinalis extracts across Caco-2 cell monolayers

Ethnopharmacological relevanceRosmarinic acid (RA), a caffeic acid-related compound found in high concentrations in Prunella vulgaris (self-heal), and ursolic acid (UA), a pentacyclic triterpene acid concentrated in Salvia officinalis (sage), have been traditionally used to treat inflammation in the mouth, and may also be beneficial for gastrointestinal health in general. Aim of the studyTo investigate the permeabilities of RA and UA as pure compounds and in Prunella vulgaris and Salvia officinalis ethanol extracts across human intestinal epithelial Caco-2 cell monolayers. Materials and methodsThe permeabilities and phase II biotransformation of RA and UA as pure compounds and in herbal extracts were compared using Caco-2 cells with HPLC detection. ResultsThe apparent permeability coefficient (Papp) for RA and RA in Prunella vulgaris extracts was 0.2±0.05×10−6cm/s, significantly increased to 0.9±0.2×10−6cm/s after β-glucuronidase/sulfatase treatment. Papp for UA and UA in Salvia officinalis extract was 2.7±0.3×10−6cm/s and 2.3±0.5×10−6cm/s before and after β-glucuronidase/sulfatase treatment, respectively. Neither compound was affected in permeability by the herbal extract matrix. ConclusionRA and UA in herbal extracts had similar uptake as that found using the pure compounds, which may simplify the prediction of compound efficacy, but the apparent lack of intestinal glucuronidation/sulfation of UA is likely to further enhance the bioavailability of that compound compared with RA.

Intestinal safety of water-soluble beta-cyclodextrins in paediatric oral solutions of spironolactone: effects on human intestinal epithelial Caco-2 cells.

Effects of water-soluble beta-cyclodextrins (beta CDs) on intestinal epithelial integrity were investigated, to establish the safe use of these beta CDs as solubilizers of spironolactone in paediatric enteral solutions. Mannitol permeability and transepithelial resistance (TER) of human intestinal epithelial Caco-2 cell monolayers during exposure to dimethyl-beta-cyclodextrin (DM beta CD), hydroxypropyl-beta-cyclodextrin (HP beta CD) and sulphobutyl ether beta-cyclodextrin (SBE beta CD) were followed. Staining methods were used to discern cells with damaged membranes and to study the integrity of cytoskeletal actin and tight junctions. Cytotoxicity of the beta CDs was tested by effects on intracellular dehydrogenase activity. Exposure to HP beta CD and SBE beta CD solutions had only minor effects on the integrity of Caco-2 cell monolayers. In contrast, DM beta CD clearly increased the epithelial permeability for the hydrophilic marker [14C]mannitol across Caco-2 monolayers, decreased TER and showed a dose-dependent cytotoxicity. According to staining, DM beta CD increased the permeability of the apical cell membrane without discernable effects on cytoskeletal actin. HP beta CD and SBE beta CD appear to be safe additives for use in enteral spironolactone preparations with respect to their acute local effects on epithelial integrity.

Intestinal transport of bis(12)‐hupyridone in Caco‐2 cells and its improved permeability by the surfactant Brij‐35

The objective of the present study was to elucidate the mechanisms of intestinal transport of bis(12)-hupyridone (B12H) to predict its oral bioavailability. The effect of the B12H concentration and the contribution of the drug efflux transporters, P-glycoprotein (P-gp or ABCB1) and multidrug resistance-associated proteins (MRPs or ABCC) on B12H absorption were measured and evaluated using the human intestinal epithelial Caco-2 cell monolayer in the presence of transporter inhibitors. The results indicated that B12H was absorbed in a dose-dependent manner at concentrations ranging from 132 to 264 µM. However, only apical efflux was observed in the directional transport studies for B12H below 88 µM (P(app) (AP-to-BL): virtually zero; P(app) (BL-to-AP): 1.591 ± 0.071 × 10(-5) cm s(-1) ). P-gp and mixed P-gp/MRP inhibitors significantly increased the absorptive transport (P(app) (AP-to-BL)) to 0.619 ± 0.018 × 10(-5) and 0.608 ± 0.025 × 10(-5) cm s(-1) , respectively, while decreasing secretory transport (P(app) (BL-to-AP)) by >75%. A multiple-MRP inhibitor, probenecid, increased the P(app) (AP-to-BL) to 0.329 ± 0.015 × 10(-5) cm s(-1) while decreasing the P(app) (BL-to-AP) by 50%. Another multiple-MRP inhibitor, indomethacin, only modestly decreased the P(app) (BL-to-AP) by ∼30% and had no effect on the absorptive transport (P(app) (AP-to-BL): virtually zero). In addition, the effect of various pharmaceutical excipients (e.g. Pluronic F-68, Tween-80 and Brij-35) on B12H transport was determined and compared. Among them, Brij-35 effectively enhanced B12H absorption at a concentration lower than its critical micelle concentration (CMC, 60 µM). Therefore, Brij-35 can be used as a potential enhancer to improve intestinal absorption of B12H for oral administration.

Kaempferol Enhances Intestinal Barrier Function through the Cytoskeletal Association and Expression of Tight Junction Proteins in Caco-2 Cells

Kaempferol, a natural flavonoid present in fruits, vegetables, and teas, provides beneficial effects for human health. We investigated the promotive effect of kaempferol on tight junction (TJ) barrier integrity in human intestinal Caco-2 cell monolayers. Transepithelial electrical resistance (TER; a TJ integrity marker) across the monolayers rapidly and markedly increased during the first 6 h after kaempferol administration and remained elevated until 48 h without any changes in the lucifer yellow or dextran fluxes. Immunoblot analysis demonstrated that kaempferol promoted the actin cytoskeletal association of the TJ proteins, zonula occludens (ZO)-1, ZO-2, occludin, claudin-1, claudin-3, and claudin-4, which was associated with the increase in TER. Kaempferol-mediated ZO-2 and claudin-4 expression was relatively smaller or occurred later than the kaempferol-promoted cytoskeletal association. Confocal microscopy showed that kaempferol-induced assembly of occludin and claudin-3 occurred at the TJ at 6 h postadministration. Extraction of cholesterol with methyl-β-cyclodextrin suppressed the kaempferol-mediated increase in TER. Sucrose density gradient centrifugation showed that the kaempferol treatment increased the TJ protein distributions in the cholesterol-rich lipid microdomain fraction. Taken together, these results indicate that the membrane lipid microdomain is involved in the kaempferol-mediated promotion of TJ protein assembly and intestinal TJ integrity.

BPR0C261 is a novel orally active antitumor agent with antimitotic and anti‐angiogenic activities

BPR0C261 is a synthetic small molecule compound cytotoxic against human cancer cells and active prolonging the lifespan of leukemia mice. In the present study, we further investigated the mechanisms of its anticancer action and found that BPR0C261 inhibited microtubule polymerization through interacting with the colchicine binding sites on tubulins, disrupted microtubule arrangement and caused cell cycle arrest at G(2)/M phase in cancer cells. BPR0C261 also inhibited the clonogenic growths of cancer cells and showed cytotoxicity against human cervical cancer cells of multidrug-resistant phenotype. In addition, BPR0C261 concentration-dependently inhibited the proliferation and migration of HUVECs and disrupted the endothelial capillary-like tube formations in HUVEC and rat aorta ring cultures. Given orally, BPR0C261 inhibited angiogenesis in s.c. implanted Matrigel plugs in mice. Notably, its IC(50) values against the endothelial cell growths were approximately 10-fold lower than those against the cancer cells. It was found orally absorbable in mice and showed a good oral bioavailability (43%) in dogs. BPR0C261 permeated through the human intestinal Caco-2 cell monolayer, suggesting oral availability in humans. Orally absorbed BPR0C261 distributed readily into the s.c. xenografted tumors in nude mice in which the tumor tissue levels of BPR0C261 were found oral dose-dependent. BPR0C261 showed in vivo activities against human colorectal, gastric, and nasopharyngeal tumors in nude mice. Most interestingly, the combination of BPR0C261 plus cisplatin synergistically prolonged the lifespans of mice inoculated with murine leukemia cells. Thus, BPR0C261 is a novel orally active tubulin-binding antitumor agent with antimitotic, apoptosis-inducing, and vasculature disrupting activities.

Permeation of Disaccharides Derived from Chondroitin Sulfate through Human Intestinal Caco-2 Cell Monolayersviathe Paracellular Pathway

The intestinal absorption of chondroitin sulfate (CS) and its hydrolysate, particularly the disaccharides (Di-CS), was investigated by using human intestinal epithelial Caco-2 cell monolayers. Although the transepithelial transport of CS was not detectable, dose- and time-dependent transport of Di-CS was observed. The transport of Di-CS was proved to be energy-independent and its permeability increased inversely with the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayers. The transport rate for Di-CS was also increased by treating the cell monolayers with such tight junction-opening agents as interferon-gamma. These results suggest that Di-CS permeated across the Caco-2 cell monolayers mainly via the paracellular pathway. The permeability of Caco-2 cell monolayers to authentic chondroitin 4-sulfate disaccharides (Di-4S), chondroitin 6-sulfate disaccharides (Di-6S) and chondroitin 0-sulfate disaccharides (Di-0S) was almost same, suggesting that sulfation did not affect the transport rate of Di-CS.

Metabolites of Galangin by 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Inducible Cytochrome P450 1A1 in Human Intestinal Epithelial Caco-2 Cells and Their Antagonistic Activity toward Aryl Hydrocarbon Receptor

Galangin, a dietary flavonoid, inhibited cytochrome P450 1A1 (CYP1A1) expression induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This inhibitory activity remained after permeating human intestinal epithelial Caco-2 cell monolayers, but was reduced when galangin permeated TCDD-pretreated Caco-2 cells. The present study tested whether TCDD affected the intestinal metabolism of flavonoids. LC-MS/MS analyses showed that galangin and two galangin glucuronoconjugates were reduced 0.7-fold, whereas kaempferol (a galangin oxidate) and kaempferol glucuronoconjugate were increased 1.5-fold by permeating TCDD-pretreated Caco-2 cells, as compared to untreated Caco-2 cells. An assay using recombinant human CYP1A1 and the CYP1A1 inhibitor alpha-naphthoflavone revealed that CYP1A1 oxidized galangin to kaempferol. These results indicated that galangin was metabolized to kaempferol by TCDD-inducible CYP1A1 in Caco-2 cells. A previous study revealed that kaempferol had much weaker inhibitory activity than galangin toward TCDD-induced CYP1A1 expression. Therefore, the oxidative metabolism of galangin to kaempferol in TCDD-pretreated Caco-2 cells implicated reduction in the inhibitory activity of galangin.

Stereoselective transport and uptake of propranolol across human intestinal Caco‐2 cell monolayers

The transport and uptake of individual propranolol (PPL) enantiomers were studied in human intestinal Caco-2 cell monolayers, and a reversed-phase HPLC-UV assay was used for quantitative analysis. S-PPL and R-PPL across Caco-2 cell monolayers was determined in the concentrations range of 10-500 microM in both apical (AP) to basolateral (BL) and BL to AP directions. S-PPL exhibited greater permeability than R-PPL in the AP to BL direction, whereas in the BL to AP direction S-enantiomer transported less than R-enantiomer. Uptake of R-PPL was significantly higher than that of S-PPL either from AP side or from BL side. The statistically significant differences in uptake were observed at the concentrations range from 10 to 50 microM. Furthermore, the apparent Michaelis constant (K(m)) and maximal velocity (V(max)) also showed significant difference between the two enantiomers. Moreover, the AP to BL transport of PPL enantiomer was markedly decreased by lowering the pH of the apical side but it did not affect the stereoselectivity of PPL across Caco-2 cell monolayers. The transport and uptake of PPL in the BL to AP direction was not influenced by several protein inhibitors. The results suggest that PPL enantiomers showed stereoselective transport and uptake across the Caco-2 cell monolayers. A special transport mechanism capable of directing the PPL enantiomers might be present in the Caco-2 monolayers.

Quercetin Enhances Intestinal Barrier Function through the Assembly of Zonnula Occludens-2, Occludin, and Claudin-1 and the Expression of Claudin-4 in Caco-2 Cells

Dietary flavonoids provide various beneficial effects for our health. We investigated the promotive effects of quercetin and myricetin on the intestinal barrier function in human intestinal Caco-2 cell monolayers. Transepithelial electrical resistance (TER) across the monolayers increased rapidly during incubation with quercetin, peaking at 6 h. Lucifer yellow flux, a paracellular marker, was dose-dependently lower after quercetin and myricetin treatments, although quercetin exhibited a more potent effect. Immunoblot analysis of tight junction (TJ) proteins revealed that zonnula occludens (ZO)-2, occludin, and claudin-1 were distributed to the actin cytoskeleton fraction by quercetin without increasing their respective whole-cell levels and this distribution was correlated with the increases in TER. The claudin-4 level was elevated by quercetin in both the cytoskeleton fraction and whole cells after 12 h. Confocal microscopy showed the assembly of claudin-1 and -4 at the TJ by quercetin. An inhibitor of protein kinase Cδ (PKCδ), rottlerin, enhanced the barrier function with changes in the distribution and expression of TJ proteins in a manner very similar to that of quercetin. Phosphorylation of PKCδ indicating the enzymatic activity in the cells was decreased by quercetin after 1 h. In the kinase assay, quercetin exhibits direct inhibition of the PKCδ isoform. This study demonstrates that quercetin enhances the intestinal barrier function through the assembly of ZO-2, occludin, and claudin-1 by inhibiting PKCδ and the increase in claudin-4 expression has an additional role after 12 h.

Transepithelial transport of ambroxol hydrochloride across human intestinal Caco-2 cell monolayers

This study aimed i) to characterize the transepithelial transport of the mucolytic agent ambroxol hydrochloride across the intestinal barrier, ii) to classify the ambroxol according to Biopharmaceutics Classification System (BCS) and iii) to predict ambroxol absorption in humans. Transport of ambroxol (100, 300 and 1000 micromol/l) was studied in a human colon carcinoma cell line Caco-2 in apical to basolateral and basolateral to apical direction, under iso-pH 7.4 and pH-gradient (6 vs. 7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+-free transport medium. Ambroxol samples from receiver compartments were analysed by HPLC with UV detection (242 nm). Results showed that ambroxol transport is linear with time, pH-dependent and direction-independent, displays non-saturable (first-order) kinetics. Thus, the transport seems to be transcellular mediated by passive diffusion. Estimated high solubility and high permeability (P(app) = 45 x 10(-6) cm/s) of ambroxol rank it among well absorbed compounds and class I of BCS. It can be expected that the oral dose fraction of ambroxol absorbed in human intestine is high.

Transport and metabolism of flavonoids from Chinese herbal remedy Xiaochaihu-tang across human intestinal Caco-2 cell monolayers1

To investigate the limiting factors for oral bioavailability of flavonoids derived from the Chinese herbal remedy Xiaochaihu-tang. The investigational flavonoids included baicalin, wogonoside, oroxylin-A 7-O-beta-Dglucopyranosiduronide, liquiritin, liquiritin apioside, isoliquiritin, and isoliquiritin apioside, as well as their aglycones baicalein, wogonin, oroxylin-A, liquiritigenin, and isoliquiritigenin. Caco-2 cell monolayers were used and the apparent permeability both in apical to basolateral and basolateral to apical directions was measured for each investigational compound. Meanwhile, chemoinformatics was carried out to provide insight into the mechanism governing the permeability. In addition, carrier-mediated transport with or without inhibitors, as well as the metabolism by conjugation, was also examined with Caco- 2 cell monolayers for the flavonoids. The investigational flavonoid aglycones exhibited favorable membrane permeability, but efficient glucuronidation and/or sulfation by the enterocytes may limit their bioavailability. For the flavonoid glycosides, their poor membrane permeability was found to be caused by high hydrogen-bonding potential. Among the glycosides, oroxylin-A 7-O-beta- D-glucopyranosiduronide, isoliquiritin, and isoliquiritin apioside were transported under the mediation of the efflux transporters multidrug resistance-associated protein and/or P-glycoprotein. The limiting factors of oral bioavailability for the flavonoids derived from Xiaochaihu-tang appeared to include poor membrane permeability, significant efflux, and efficient intestinal metabolism by conjugation.

Intestinal Transport of Free Anthraquinones in Caco-2 Cell Model

AIM: To investigate the transepithelial transport of the six free anthraquinones 1,8-dihydroxyanthraquinone (DQ),chrysophanol (CP),emodin (EN),rhein (RN),aloe-emodin (AE) and citreorosein (CN) in the human intestinal Caco-2 cell monolayer model. METHODS: The monolayers of the human intestinal epithelial cancer cell line Caco-2 were incubated with DQ,CP,EN,RN,AE and CN to model its intestinal absorption and transport,respectively. The determination of the compounds was performed by high-performance liquid chromatography. RESULTS: The apparent permeability coefficients (Papp) in the Caco-2 cell monolayers for DQ,CP,EN,RN,AE and CN were (2.16±0.38)×10-6,(3.00±0.09)×10-7,(1.99±0.94)×10-6,(4.58±0.20)×10-6,(6.24±0.19)×10-6 and (7.34±0.61)×10-6 cm ·s-1 from the apical-to-basolateral direction,respectively,and (3.20±0.10)×10-6,(4.50 ± 0.02)×10-7,(2.22 ± 0.62)×10-6,(4.94 ± 0.39)×10-6,(2.87 ± 0.18)×10-6 and (6.80 ± 0.37)×10-6 cm ·s-1 from the basolateral-to-apical direction,respectively. The Papp values of DQ,EN,RN,AE and CN were about 10 times less than those (6.30 ± 0.26)×10-5 cm ·s-1 of propranolol as a reference standard compound of high permeability and the main mechanism of intestinal absorption by passive diffusion,and about 10 times higher than those ( 1×10-7 cm ·s-1) of atenolol as a reference standard compound of poor permeability and the main mechanism of intestinal absorption by passive diffusion. Whereas,the Papp value of CP was at nearly the same magnitude with those of atenolol. In the presence of iodoacetamide,the Papp of AE were not affected in both apical-to-basolateral and basolateral-to-apical directions. CONCLUSION: The intestinal absorption of all test compounds was passive diffusion as the dominating process in Caco-2 cell monolayer model. DQ,EN,RN,AE and CN will be moderately absorbed compounds and CP was estimated to be a poorly absorbed compound.

High‐ and low‐capacity taurine transport across the luminal membrane of the small intestinal epithelium

Taurine is a “semi‐essential” beta‐amino acid; yet it is not clear which transporter(s) mediates taurine uptake across the small intestinal luminal membrane. The three intestinal beta‐amino acid transporters [taurine transporter TauT (SLC6A6), proton‐coupled amino acid transporter PAT1 (SLC36A1), and ATB0,+ transporter (SLC6A14)] were expressed in Xenopus laevis oocytes, and radiolabeled β‐alanine or taurine uptake characterized. TauT and PAT1 both transport taurine and β‐alanine whereas ATB0,+ transports only β‐alanine. To determine the relative contribution of each carrier to small intestinal transport, β‐alanine and taurine uptakes were measured across the apical membrane of human intestinal Caco‐2 cell monolayers. Use of selective inhibitors and ion substitutions identified roles for PAT1 and TauT (but not ATB0,+) in small intestinal uptake of these beta‐amino acids (ATB0,+ function may be more important in the colon). Under physiological conditions (apical pH 6.5, presence of Na+), the relative contributions of PAT1 and TauT to taurine uptake were dependent upon the taurine concentration. The high‐capacity, low‐affinity PAT1 may provide the major route for small intestinal taurine absorption from taurine‐rich foods whereas the low‐capacity, high‐affinity TauT may scavenge small quantities of taurine from the lumen. Supported by The Wellcome Trust (grant number 078640/Z/05/Z).

Transepithelial Transport of α-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.

TCDD-Induced CYP1A1 Expression, an Index of Dioxin Toxicity, Is Suppressed by Flavonoids Permeating the Human Intestinal Caco-2 Cell Monolayers

Since the toxicological effects of dioxins are mainly mediated by the aryl hydrocarbon receptor (AhR), an in vitro assessment system for AhR activity was used in this study to search for flavonoids that attenuated dioxin toxicity through the intestinal epithelial monolayer. When AhR transformation in Hepa-1c1c7 cells was examined by southwestern ELISA, nine flavonoids among 34 kinds of flavonoids inhibited the transformation by more than one-half. When each flavonoid with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was added to dioxin-responsive HepG2 cells, seven flavonoids significantly restrained the TCDD-induced transcriptional activity of the CYP1A1 promoter. Furthermore, those seven flavonoids that had permeated the Caco-2 cell monolayers demonstrated an inhibitory effect on both the AhR transformation and on the transcriptional activity of the CYP1A1 promoter. The expression level of the CYP1A1 mRNA and protein induced by TCDD was suppressed by flavone, galangin, and tangeretin. It is proposed from these results that some flavonoids have the ability to suppress dioxin-induced AhR activity after permeating the human intestinal epithelial cell monolayer.

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.

Transport, deglycosylation, and metabolism of trans-piceid by small intestinal epithelial cells

Numerous epidemiological and animal studies have shown that consumption of red wine is related to reduced incidence of cardiovascular diseases and cancer. Trans-resveratrol (3, 5, 4'-trihydroxystilbene), a phenolic compound present in wine, has been reported to have a potential cancer chemopreventive activity. Moreover, it may exert a protective effect against atherogenesis through its antioxidant properties. Trans-piceid (3-ss glucoside of trans-resveratrol) is present to a greater extent than its aglycone in red wine, but hydrolysis of this glycosylated derivative can occur in small intestine and liver, which would enhance the amount of the biological active trans-resveratrol. The present study aimed to investigate the rate of transepithelial transport of trans-piceid using human intestinal Caco-2 cell monolayers and metabolism of this compound during its absorption across the small intestine. The transport of trans-piceid was evaluated in the human epithelial cell line Caco-2, which possesses enterocyte-like properties in vitro. For transepithelial experiments, confluent monolayers of Caco-2 cells were grown on Transwell inserts. For metabolic studies, we used both Caco-2 cells seeded on 6-well plates and rat small intestine cell-free extracts. The time course of apical (AP) to basolateral (BL) transport of trans-piceid showed that the favorable apparent permeability coefficient (Papp) declined rapidly during the 6 h of the experiment. This observation could be correlated with the appearance of metabolites. After incubation of Caco-2 cells with trans-piceid, trans-resveratrol was detected on both AP and BL sides. By using protein extracts obtained from rat, we conclude that the Lactase Phlorizin Hydrolase (LPH) and Cytosolic-ss-Glucosidase (CBG) are involved in the hydrolysis of trans-piceid. Furthermore, we show that after deglycosylation, the resulting aglycone is metabolized in trans-resveratrol-3-O-ss-glucuronide and to a lesser extent in trans-resveratrol-4'-O-ss-glucuronide, and that UGT1A1 is mainly involved in this metabolism. This study demonstrates that the transepithelial transport of trans-piceid occurs at a high rate and that the compound is deglycosylated in trans-resveratrol. There are two possible pathways by which trans-piceid is hydrolyzed in the intestine. The first is a cleavage by the CBG, after passing the brush-border membrane by SGLT1. The second is deglycosylation on the luminal side of the epithelium by the membrane-bound enzyme LPH, followed by passive diffusion of the released aglycone, which is further metabolized inside the cells into two glucuronoconjugates.

Lack of dextromethorphan interaction with P-glycoprotein

Background The analgesic anti-NMDA effect of dextromethorphan (DEM) might result mainly from a central action of unchanged DEM rather than from its more active metabolite dextrorphan (DOR). This could be related to a weaker interaction of DEM with the efflux drug transporter P-glycoprotein (P-gP) as compared with that of DOR. Aim We studied the involvement of P-gP in the transepithelial transport of DEM and DOR. Methods We used the human intestinal Caco-2 cell monolayer model. Bidirectional transport of 1, 5 and 10 μM DEM across the Caco-2 monolayers was investigated in the presence and absence of the P-gP inhibitor verapamil (100μM). DEM quantitation was performed using HPLC. Results We found that: i) DEM showed no statistically significant differential transport rates between the basolateral-to-apical (B-A) and apical-to-basolateral (A-B) directions at 5 and 10μM (apparent permeability coefficient Papp: 17 vs 16.10−6 and 29 vs 31.10−6 cm/s respectively). However, at 1μM, the rate of transport A-B was greater (Papp: 5 vs 7.10−6 cm/s); ii) B-A transport of DEM was not affected by verapamil (Papp: 5 vs 5.10−6; 17 vs 16.10−6 and 29 vs 30.10−6 cm/s); iii) The transepithelial transport of DEM was concentration-dependant. Conclusion The data indicate that DEM is not a P-glycoprotein substrate at therapeutic concentrations and that the mechanism of diffusion is passive. Clinical Pharmacology & Therapeutics (2005) 77, P10–P10; doi: 10.1016/j.clpt.2004.11.039

Transepithelial Permeability of Myricitrin and Its Degradation by Simulated Digestion in Human Intestinal Caco-2 Cell Monolayer

Myricitrin permeated the human intestinal Caco-2 cell monolayer via the paracellular pathway in a time- and concentration-dependent manner. Myricitrin was not conjugated by Caco-2 cells. Myricitrin was degraded by simulated intestinal digestion, but permeability did not change significantly.