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

## TL;DR

Punicalagin, a polyphenol in pomegranates, blocks gut bacteria from converting l-carnitine into harmful trimethylamine (TMA), which is linked to heart disease.

## Contribution

This study identifies punicalagin as the specific pomegranate polyphenol that inhibits TMA production from l-carnitine in a human colon model.

## Key findings

- Punicalagin significantly inhibited the conversion of l-carnitine to γ-butyrobetaine and TMA.
- Punicalagin almost completely blocked TMA production compared to the control.
- Pomegranate extract lowered pH and inhibited l-carnitine metabolism, suggesting acidification may also play a role.

## Abstract

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.

Punicalagin is the single pomegranate polyphenol inhibiting the production of proatherogenic trimethylamine (TMA) from l-carnitine in vitro. Dietary l-carnitine is converted by gut microbiota to TMA. Created in BioRender.