Study On The Transport And Mrtabolism Of Catechins Across Caco-2 Monolayer Models

Tea polyphenols are the most important functional components of tea,among which about 70%to 80%of the total amount are catechins.A series of pharmacological effects of catechins have been reported,including anti-oxidative activity,anti-mutation activity,and disinfection of antimicrobial and preventive effects of many diseases such as cancer,cardiovascular disease,and diabetes and so on.In this research,the bidirectional transport and metabolism of eight green tea catechins were studied in Caco-2 monolayers aiming to investigate the bioavailability and biotransformation and their molecular mechanism of catechins.It provide theoretical basis for finding ways to increase the oral bioavailability so as to improve the pharmacological effects of catechins in vivo.The main research contents and results are as follows:1.The establishment of Caco-2 cell monolayersIn this research,the transepithelial electrical resistance(TEER),alkaline phosphatase activity and fluxes of Lucifer yellow were measured to evaluate and monitor the integrity,the cell polarity and the permeability of Caco-2 cell monolayer.The results showed that the Caco-2 monolayers in our lab could meet the needs of the experiments and the criterion of Caco-2 monolayers were established as followed:TEER should be more than500?·cm²(12-well transwell plate or 6-well transwell plate);the Papp value of Lucifer yellow should be less than 0.5×10^-6cm/s;and the activity of alkaline phosphatase AP/BL should be more than 5.With this criterion the monolayers could be used for transport and metabolism experiments.2.The bidirectional transport of catechins in Caco-2 monolayers and their effects on the related transporters in Caco-2 cellsCaco-2 cell monolayer was used as an in vitro model to study the possible absorption and transport mechanism of catechins in the small intestine.The results showed that catechin(C),gallocatechin(GC),catechin gallate(CG)and gallocatechin gallate(GCG),epicatechin(EC),epigallocatechin(EGC),epicatechin gallate(ECG)and epigallocatechin gallate(EGCG)could all penetrate the monolayer of Caco-2 cells directly but their apparent permeability coefficients were all less than 1×10⁶ cm/s,suggesting a poor oral bioavailability of catechins.The flux from apical side(AP side)to the basolateral side at the concentration from 200?mol/L to 400?mol/L were all linear for up to 2 h in a concentration dependent manner(r²?0.96),which indicated that the passive transport was the main pathway for catechins.The BL to AP flux are increased with increasing concentration with saturation at concentrations higher than 300?mol/L.The amount of efflux transport was significantly greater than that of absorption transport,and the efflux ratio(ER)of catechins were all larger than 2.Consistently,it indicated that the permeation mechanism of catechins may be passive diffusion with a BL to AP active efflux involved.MK-571,an inhibitor of multidrug resistance protein 2(MRP2),was used in transport of catechins in Caco-2 cell monolayers.The results showed that MK-571could inhibit the efflux of catechins and increase absorption transport of catechins,indicating that catechins were the substrates of MRP2.The biderectional transport of cis(epi)catechins(EC,ECG,EGC,EGCG)and trans catechins(C,CG,GC,GCG)were compared.The amount transported of trans catechins are higher than the amount of their corresponding cis(epi)catechins at each concentration and each time point tested.It indicated that stereochemical characteristics may be a crucial factor with regard to catechins absorption in humans.After incubation with catechins,the m RNA level was tested by q RT-PCR and the protein level was examined by Western blotting.The results showed that all catechins could up-regulate the expression of MRP2 at the transcriptional and translation level(p<0.05).The increasing expression of MRP2 can also increase the efflux of catechins which make an even lower bioavailability of tea catechins.Besides,catechins are selective in regulating the transcriptional and translation levels of multidrug resistance protein 1(MRP1).C,EC,EGCG are more likely to regulate the expression of MRP1 at the transcriptional and translation level(p<0.05).In addition,the effect of catechins on the expression of glucose transporters was analyzed.The results showed that both the m RNA and protein level of sodium-glucose co-transporter 1(SGLT1)were significantly down-regulated after incubation with catechins(p<0.05)in Caco-2 cells.The decrease expression of SGLT1 may lead an increasing absorption of glucose.On the other hand,expression of glucose transporter 2(GLUT2)decreased at transcriptional and translation levels with a short time incubation(2 h)while the expression of GLUT2 increased after prolonging incubation time to 24 h.3.The metabolism of catechins in Caco-2 cells monolayerThe metabolism of catechins in Caco-2 cell monolayer was studied by LC/MS (negative mode)and LC/MS/MS techniques.The results showed that the m/z of the metabolites was small(less than 650),indicating that the metabolism of catechins in intestinal epithelial cells was mainly simple metabolism.By comparing the relative contents,the sulfated metabolites of the eight catechins were found to be the most abundant.Therefore,sulfation is the most important pathway for catechins in intestinal epithelial cells.Secondly,the methylation metabolites of catechins were also at a higher level.With many peaks in LC chromatogram,it indicated that there were many isomerides existing.Methylated sulfate conjugates were only observed in the efflux transport of CG,GC,EGC and GCG.Comparing the relative contents of metabolites between cis(epi)catechins and trans catechins,we found that trans catechins had a more extensive metabolism in Caco-2monolayers than cis catechins when transported from BL to AP.In addition,there are some differences in the metabolites between absorption transport and efflux transport.The samples in receiver chambers of the AP to BL transport with non-gallated catechins(C EC GC and EGC)showed the same types of metabolites formed as that in the apical samples from the BL to AP transport,but with much fewer amount than the later one.However,in absorption transport of gallated catechins(CG ECG GCG and EGCG),none of the sulfation or methylation metabolites were found.It indicates that the metabolites of gallated catechins are more difficult to be absorbed by intestinal tract.Therefore,the extensive metabolism of catechins is another important factor limiting the oral bioavailability of catechins.