In vitro stability and ex vivo absorption of thymol monoglucosides in the porcine gut

Abstract

Thymol α-D-glucopyranoside (TαG) and thymol β-D-glucopyranoside (TβG) are believed to have different kinetic behaviours in the porcine gut than its parent aglycon thymol. However, recently, it was shown that concentrations of both glucosides decreased rapidly in the stomach and proximal small intestine following oral supplementation to piglets as did thymol. Yet, the stability of thymol glucosides in gut contents and their absorption route remains obscure. Therefore, a series of in vitro incubations were performed, simulating the impact of pH, digestive enzymes, bacterial activity and mucosal extracts on stability of these glucosides. Their absorption mechanisms were investigated using the Ussing chamber model in the presence or the absence of inhibitors of sodium-dependent glucose linked transporter 1 and lactase phlorizin hydrolase. Both glucosides remained intact at physiological pH levels in the presence of digestive enzymes. Recoveries from TαG and TβG were below 90% when incubated with small intestinal homogenates from the distal jejunum or from all sampled sites, respectively. However, no aglycon could be detected in these samples. Bacterial inoculum of the small intestine, on the other hand, hydrolysed TβG quickly with up to 44% of free aglycon appearing. TαG proved more resistant to porcine gastro-intestinal bacterial glucosidases with only trace amounts (<1%) of free thymol at the end of the incubations. Electrophysiological measurements in Ussing chambers did not suggest active transport of the glucosides. Mucosal TαG and TβG concentrations were unchanged between start and end of the absorption measurements. Additionally, no TαG and only a very limited amount of TβG were retrieved from the serosal side. Tissue associated concentrations, although marginal (<1% of luminal concentration), were mainly as intact glucoside or as aglycon for TαG and TβG, respectively. Addition of both inhibitors significantly increased the amount of intact glucosides retrieved from the mucosal tissues as compared to controls. In conclusion, bacterial hydrolysis was identified as the most important source of TβG loss, whereas TαG seemed less prone to degradation or absorption in these in vitro and ex vivo models.