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Metabolic products of choline promote atherosclerotic cardiovascular disease.Trigonelline was isolated, purified and characterized from hydroalcoholic extract of Trigonella foeneum graecum seeds.A gram negative anaerobic bacteria, Citrobacter freundii, was isolated from gut microbiota.Citrobacter freundii is responsible for choline metabolism and trigonelline inhibit the activity of C. freundii.Trigonelline decreased TMAO levels in mice by inhibiting C. freundii and FMO3 enzyme.Gut microbiota based metabolism of choline produces trimethylamine (TMA) which is further converted to a pro-atherosclerotic metabolite, trimethylamine-N-oxide (TMAO) by flavin monooxygenase (FMO3). Trigonelline from the plant Trigonella foenum-graecum has been reported for the treatment of CVD. Aim of the present study was to check the effect of trigonelline on the gut microbiota based conversion of TMA to TMAO. Trigonelline was isolated from hydroalcoholic extract of seeds of Trigonella foenum-graecum. The isolated trigonelline was characterized through TLC and UPLC-MS. Anaerobic microbe responsible for the metabolism of choline to TMA was isolated by culturing the human gut microbiota in choline enriched medium. The isolated bacteria was identified at molecular level based on PCR amplification of 1500bp of 16S rRNA gene sequence. Isolated FMO3 was used for ex vivo conversion of TMA to TMAO. Further, we investigated the effect of trigonelline in isolated gut microbe based metabolism of choline, lipid profile and TMAO levels in mice with or without suppression of gut microbiota with antibiotics. Liquid-liquid purification and chromatographic analysis confirmed the trigonelline purity (87.26%) and which was also confirmed by mass spectroscopy with m/z 137.4 in positive ionization mode. A total of 30 anaerobic microbes responsible for TMA production were isolated and Citrobacter freundii was the superior among others for the production of TMA. In vitro culture of C. freundii in choline enriched medium supplemented with trigonelline resulted in significantly reduction TMA and followed by TMAO production. In ex vivo, a maximum of 85.3% TMAO production was reduced by trigonelline at concentration of about 300 μg/mL. Serum level of lipids and TMAO were significantly altered in choline fed animals with or without suppression of gut microbiota and this phenomenon was reversed upon the oral administration of trigonelline in a dose-dependent manner. This study demonstrates the effect of trigonelline on gut microbiota responsible for choline metabolism and this can be used as a model for evaluation of herbal drugs and its effect in gut microbiota prompted cardiovascular disorders.