Punicalagin is the key pomegranate polyphenol inhibiting gut microbial trimethylamine (TMA) production from L-carnitine in an in vitro human colon model.

TMAO has been linked to various cardiometabolic diseases and all-cause mortality risk. A major dietary precursor of TMAO is L-carnitine. L-Carnitine is metabolised by microbiota to γ-butyrobetaine (γ-BB), followed by trimethylamine (TMA), and is then oxidised to TMAO in the liver. Previously, we have shown that a polyphenol-rich pomegranate extract dose-dependently inhibited the production of γ-BB and TMA from L-carnitine. Here, we further investigated the effects of the pomegranate extract and its individual constituents/metabolites (polyphenols, spray-drying agent gum Arabic, and urolithins) on the microbial metabolism of L-carnitine to γ-BB and TMA using a high-throughput in vitro model of the human colon. A small-scale, high-throughput colon model was inoculated with L-carnitine, individual constituents of the extract (2 mg mL-1), and 1% human faecal inoculum, while continuously monitoring pH. Samples were collected over 48 hours, and methylamines were quantified using LC-MS/MS with isotopically labelled internal standards. Punicalagin, but not the other constituents, inhibited the conversion of L-carnitine to γ-BB (p < 0.001) and almost completely blocked TMA production compared to the control (p < 0.003). Furthermore, including the whole pomegranate extract in the high-throughput colon model significantly reduced the pH and completely inhibited L-carnitine metabolism, suggesting that acidification may also inhibit microbial L-carnitine metabolism. Here it was shown that, of all the tested phenolic and non-phenolic components of the pomegranate extract, only punicalagin inhibited TMA production from L-carnitine, highlighting it as a promising inhibitor of TMA and potentially TMAO formation.