We investigated the effect of theaflavins (TFs) on membrane barrier of Caco-2 cells. For fluorescein-transport experiments, the apparent permeability (Papp) of fluorescein in Caco-2 cells pretreated with 20 μM TFs were significantly decreased compared with that in untreated cells. Although the respective monomeric catechins did not show any Papp reduction, purpurogallin pretreatment resulted in a significant Papp reduction similar to that of TF-3'-O-gallate (TF3'G) pretreatment. This indicates that the benzotropolone moiety may play a crucial role in the Papp reduction or tight junction (TJ)-closing effect induced by TFs. In TF-3'-O-gallate-pretreated Caco-2 cells, fluorescein transport was completely restored by compound C (AMPK inhibitor). In addition, TF3'G significantly increased both the mRNA and protein expression of TJ-related proteins (occludin, claudin-1, and ZO-1) as well as the phosphorylation of AMPK. It was, thus, concluded that TFs could enhance intestinal barrier function by increasing the expression of TJ-related proteins through the activation of AMPK in Caco-2 cells.

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There is no analytical system to directly and simultaneously detect taste- and odor-active compounds. Graphite carbon black (GCB) nanoparticles, a chemical adsorbent for a variety of compounds, including gaseous molecules, which consists of nanoparticles made from multilayered sheets of sp2-conjugated atomic carbon, have the characteristics required for a surface-assisted laser desorption ionization–mass spectrometry (SALDI-MS) material. Thus, in this study, GCB-LDI-MS was developed for the simultaneous detection of taste- and odor-active compounds. The proposed GCB-LDI-MS successfully detected volatile compounds, including ethyl esters, alcohols, fatty acids, and aldehydes with up to 16 carbons (maximum carbon number of the tested compounds), which could not be detected by conventional matrix-assisted-LDI-MS. Moreover, a series of taste-active compounds (e.g., amino acids and sugars) and various volatile compounds (e.g., ethyl esters, aldehydes, alcohols, fatty acids, and lactones) as a mixture in liquid samples were also detected by the present GCB-LDI-MS. Thus, the proposed simple and rapid detection technique can digitize the analysis of flavor compounds, which provides an analytical concept for flavor scanning to evaluate the palatability of food products.

Objective: In the small intestine, peptide transporter 1 (PEPT1) plays a role in the transport of di- and tri-peptides. Recently, we found that theaflavins (TFs), dimeric catechins, inhibited the transport of di-peptides across Caco-2 monolayers by suppressing the expression of PEPT1 through AMP-activated protein kinase (AMPK) activation. In this study, we investigated the structural requirement of theaflavins for the effect, and the mechanism(s) underling theaflavin-induced AMPK activation.Methods: Theaflavin-3’-O-gallate (TF3’G) was used for this study, since it possessed the most potent inhibition power for peptide-transport among theaflavins. Absorption ability was measured with Caco-2 cell monolayers treated with or without 20 μM sample (TF3’G or its related compounds) in an Ussing Chamber. The amount of Gly-Sar (a model of PEPT1-transporing peptide) transport at fixed time-points to 60 min was determined by fluorescent naphthalene-2,3-dicarboxaldehyde-derivatized assay (Ex/Em: 405 nm/460 nm). The apparent permeability coefficient (Papp) was used to evaluate the permeability. Expression of PEPT1 protein in Caco-2 cells treated with or without 20 μM TF3’G in the presence or absence of inhibitor (10 μM compound C as AMPK inhibitor or 25 μM STO-609 as CaMKK inhibitor) was evaluated by Western blot.Results: The Papp value of Gly-Sar significantly (P < 0.05) decreased in 20 μM purprogallin-treated Caco-2 cells as well as in TF3’G-treated cells, together with the reduction of PEPT1 expression, while their monomeric catechins did not show any Papp reduction. In TF3'G-treated Caco-2 cells, the recovery of the reduced PEPT1 expression was found by 10 μM compound C, but not STO-609.Conclusion: The study demonstrated that the benzotropolone moiety in theaflavins was a crucial structural requirement for exerting the inhibition of intestinal peptide-transport, and the suppression of PEPT1 expression by theaflavins would be caused by activating LKB1/AMPK pathway, but not CaMKK/AMPK pathway.Keywords: Theaflavin-3’-Ο-gallate, Peptide transport, PEPT1, Benzotropolone, AMP-activated protein kinase, Calmodulin-dependent protein kinase kinase

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