Atherosclerosis amelioration by allicin in raw garlic through gut microbiota and trimethylamine-N-oxide modulation

Abstract

Cardiovascular disease (CVD) is strongly associated with the gut microbiota and its metabolites, including trimethylamine-N-oxide (TMAO), formed from metaorganismal metabolism of ʟ-carnitine. Raw garlic juice, with allicin as its primary compound, exhibits considerable effects on the gut microbiota. This study validated the benefits of raw garlic juice against CVD risk via modulation of the gut microbiota and its metabolites. Allicin supplementation significantly decreased serum TMAO in ʟ-carnitine-fed C57BL/6 J mice, reduced aortic lesions, and altered the fecal microbiota in carnitine-induced, atherosclerosis-prone, apolipoprotein E-deficient (ApoE-/-) mice. In human subjects exhibiting high-TMAO production, raw garlic juice intake for a week reduced TMAO formation, improved gut microbial diversity, and increased the relative abundances of beneficial bacteria. In in vitro and ex vivo studies, raw garlic juice and allicin inhibited γ-butyrobetaine (γBB) and trimethylamine production by the gut microbiota. Thus, raw garlic juice and allicin can potentially prevent cardiovascular disease by decreasing TMAO production via gut microbiota modulation.

Conflict of interest statement

The authors declare no competing interests.

© 2022. The Author(s).

Figures

Fig. 1. Allicin reduced serum TMA, TMAO, and γBB levels, and ʟ-carnitine principally changed the fecal microbiome composition in ʟ-carnitine-fed male C57BL/6 J mice (n = 10 per group).

a Experimental design; b serum TMA; c serum TMAO; d serum γBB; e principal coordinate analysis (PCoA) plot with Bray−Curtis dissimilarity; f observed OTUs α-diversity indices; and g relative abundance of fecal microbiota at family level. Dot plots are expressed as the mean ± SD or median; statistical analyses of serum bacterial metabolites were performed using the Wilcoxon signed-rank test Con vs. Con + A group, Con vs. Carn group; Kruskal–Wallis tests with Dunn’s multiple comparisons, Carn vs. Carn + A group, Carn vs. Carn + D group, and Carn + A vs. Carn + D group. Gut microbiota ɑ-diversity was determined using the unpaired two-tailed Student’s t-test; one-way ANOVA with Tukey’s range test. The statistical analyses of the relative abundance were performed using the unpaired Wilcoxon signed-rank test with the false discovery rate (FDR), Con vs. Con + A group (#P < 0.1); Con vs. Carn group (*P < 0.1); Carn vs. Carn + A group (†P < 0.1); Carn + D group (‡P < 0.1).

Fig. 2. Raw garlic juice reduced plasma TMAO formation ability, increased plasma γBB level in healthy TMAO producers, and shaped fecal microbiota composition via increasing the evenness, ɑ-diversity, and relative abundance of specific beneficial bacterial taxa.

a Experimental design, healthy participants (n = 9) were screened for TMAO production using oral carnitine challenge test (OCCT), the criterion for categorization as high-TMAO producers was plasma TMAOMAX > 10 μM. High-TMAO producers (n = 7) subsequently received an intervention of garlic juice (55 mL, equivalent to 48 mg of allicin/day) for 1 week, followed by OCCT; b plasma and c urine TMAO, TMAOMAX, and TMAOAUC; d plasma γBB and γBBAUC. e Principal coordinate analysis (PCoA) plot with Bray−Curtis dissimilarity; f Shannon α-diversity index; g family-level composition of fecal microbiome; h volcano plot of fecal microbiota before and after garlic juice intervention; and i heatmap of the relative abundances of fecal microbiota with a significant difference using the Wilcoxon signed-rank test (P < 0.05). Data are expressed as the mean ± SEM; statistical analyses were performed by using the two-tailed paired Student’s t-test (*P < 0.05; **P < 0.01; and ***P < 0.001). The relative abundance bar plot statistical analysis was performed using a paired Wilcoxon signed-rank test (*P < 0.05). Volcano plot: red points indicate OTUs with a P-value < 0.05 and log2 fold-change > 1; orange points indicate OTUs with a P-value < 0.05; and blue points indicate log2 fold-change > 1.

Fig. 3. Allicin reduced aortic lesions through the reduction of TMA and TMAO formation and changed the fecal microbiome composition in ʟ-carnitine-induced atherosclerosis female ApoE−/− mice (n = 9 per group).

a experimental design; b representative image of oil red-stained enface aorta, scale bar is 0.5 cm; c percentage of aortic lesion; d d9-TMA level according to oral carnitine challenge test (OCCT) and its AUC; e d9-TMAO level of OCCT and its AUC; f d9-γBB level according to OCCT and its AUC. g α-diversity indices, observed OTUs, Shannon index, and Chao1 index; h Principal coordinate analysis (PCoA) plot with Bray−Curtis dissimilarity; and i heatmap of the relative abundances of fecal microbiota with a significant difference using the Kruskal–Wallis test with false discovery rate (FDR) (P < 0.001). OCCT curves are expressed as the mean ± SEM, and dot plots are expressed as the mean ± SD; Statistical analyses were performed using an unpaired two-tailed Student’s t-test Con vs. Con + A group, Con vs. Carn group; one-way ANOVA with Tukey’s range test for comparisons Carn vs. Carn + A group, Carn vs. Carn + D group, and Carn + A vs. Carn + D group.

Fig. 4. Garlic-juice and allicin inhibited the formation of d9-γBB and d9-TMA in vitro after inoculation of TMA/γBB-producing bacteria and ex vivo after inoculation of human feces from high-TMAO producers.

a inhibitory effect of garlic juice and allicin on the bacteria converting carnitine → γBB (co-culture of Proteus penneri, Escherichia fergusonii, and Edwardsiella tarda) in Wilkins–Chalgren (WC) broth supplemented with d9-carnitine; b inhibitory effect of garlic juice and allicin on the bacteria converting γBB to TMA (Emergencia timonensis) in WC broth supplemented with d9-γBB. c Inhibitory effect of garlic juice and allicin on the high-TMAO-producing gut microbiota in WC broth supplemented with d9-carnitine and d d9-γBB. The utilization of d9-carnitine/d9-γBB and the production of d9-γBB/d9-TMA were measured at 0, 6, 12, and 24 h. Half-maximal inhibitory concentration (IC50) values were determined using nonlinear regression (see supplementary Fig. 13).

Fig. 5. Effect of allicin and raw garlic juice on CVD prevention and atherosclerosis amelioration through gut microbiota and TMAO modulation.

(1) Allicin decreased the gut microbiota- and host-derived TMA and TMAO levels in mice supplemented with ʟ-carnitine in drinking water; (2) Raw garlic juice reduced the plasma and urine TMAO levels and increased the beneficial gut bacterial abundance in high-TMAO producing humans; (3) Allicin ameliorated aortic lesions by reducing the TMA/TMAO production and altered the gut microbiome in ʟ-carnitine-induced atherosclerotic ApoE−/− mice; (4) Raw garlic juice and allicin inhibited microbial pathway of carnitine → γBB → TMA conversion in specific bacteria and high-TMAO producer’s gut microbiota.