Effect Of Milk On Catechins In Vitro Simulated Digestion And Caco-2Cell Monolayer Transport

Catechins are abundant in tea shoots, accounting for12%～24%of the dry weight of tea leaves and are believed not only play an important role in the formation of tea color, smell and taste, but also as the primary components in tea functional ingredients. Digestive release from the food or beverage matrix and then absorbed by intestinal cells is the foundation of catechins to play health function. In vivo feeding methods, using animals or humans, are operational complexity, time consuming and poor repeatability, which is why more and more researchers are using in vitro models to mimic digestion and absorption of nutrients or drugs. In this study, the in vitro physiological conditions of gastrointestinal tract considering enzymes, pH and temperature were established to digest tea and tea with milk, then the effect of milk concentration, pH, digestive enzymes and temperature on the recoveries of tea catechins were comprehensively evaluated. In addition, the in vitro Caco-2cell model, which possesses the similar morphological and functional properties to the human intestinal epithelial cells were also established. Digestive juice was loaded on the apical side of monolayer and the transported catechins were received from basolateral side. The transport experiments provide a clue to investigate the impact of milk components on the catechins intestinal absorption. Results of this study showed that:1. Milk proteins can bind to catechins and form the catechin/protein complexes. After adding10%milk into tea solutions, the recovery of EGCG was decreased to41.0%. An inverse association was showed in the mixture with milk concentration increased, the free catechins decreased. Gallated catechins exhibited the stronger association with milk proteins than non-gallated catechins. While during simulated gastric digestion, catechins were quite stable with only slightly decrease of EGCG and ECG. Mix10%milk, the recovery of EGCG was83.5%. During simulated intestinal digestion, all catechins except EC were decreased. EGCG showed the largest degradation with a68.6%recovery when the milk concentration was at10%.2. Factors such as pH values, digestive enzymes and brewing temperatures do impact the binding between catechins and proteins. This paper showed that the higher the pH, the more unstable the catechins. Digestive enzymes also affected the stability of catechins at the same pH value as the results showed that group with digestive enzymes obtained more catechins. In addition, the content of catechins in hot GTE solutions brewed at a temperature of100℃compared to23℃was decrease7.2%, followed with lower recoveries after addition of milk. Surprisingly, the results showed that catechins in hot GTE solutions obtained higher recoveries after simulated gastric and intestinal digestion.3. Stability of Catechins in HBSS buffer under cell culture condition showed that compared to the aqueous solution, catechins were unstable in HBSS buffer at both pH6.5and7.4, especially EGCG with a loss of29%and43%, respectively. The observation of catechins stability in HBSS buffer at pH6.5and7.4in the course of time showed timedependent degradation of EGC and EGCG. A30%and29%loss of EGC and EGCG, respectively, was observed during incubation in HBSS at pH6.5after2h. Both compounds were even more labile in HBSS at pH7.4, the loss of52%and44%, for EGC and EGCG, respectively was observed after2h. The other catechins, i.e. C, EC, GCG and ECG were quite stable in HBSS buffers with slight changes during2h incubation.4. The cytotoxicity measurement showed that green tea extracts (GTE) at high concentration had a damage effect to Caco-2cells with IC50values of8.32mg/mL, while the concentration at1.00mg/mL performed in this study had no significant damage to Caco-2cells. The transport experiment revealed that despite higher content of available catechins in GTE and GTE digest comparing to GTE+10%and GTE+25%milk digests, the transported catechins was noted only for tea preparations with milk. In the sample GTE+10%milk digest, the highest Papp value of7.28×10-6cm/s was obtained for GCG, followed by EGC, EGCG, ECG and EC What’s more, recoveries of catechins in Caco-2cells after2h incubation also increased with milk addition.