Flavonoids in Amomum tsaoko Crevost et Lemarie Ameliorate Loperamide-Induced Constipation in Mice by Regulating Gut Microbiota and Related Metabolites

Abstract

Amomum tsaoko (AT) is a dietary botanical with laxative properties; however, the active ingredients and mechanisms are still unclear. The active fraction of AT aqueous extract (ATAE) for promoting defecation in slow transit constipation mice is the ethanol-soluble part (ATES). The total flavonoids of ATES (ATTF) were the main active component. ATTF significantly increased the abundance of Lactobacillus and Bacillus and reduced the dominant commensals, such as Lachnospiraceae, thereby changing the gut microbiota structure and composition. Meanwhile, ATTF changed the gut metabolites mainly enriched in pathways such as the serotonergic synapse. In addition, ATTF increased the serum serotonin (5-HT) content and mRNA expression of 5-hydroxytryptamine receptor 2A (5-HT_2A), Phospholipase A2 (PLA2), and Cyclooxygenase-2 (COX2), which are involved in the serotonergic synaptic pathway. ATTF increased Transient receptor potential A1 (TRPA1), which promotes the release of 5-HT, and Myosin light chain 3(MLC3), which promotes smooth muscle motility. Notably, we established a network between gut microbiota, gut metabolites, and host parameters. The dominant gut microbiota Lactobacillus and Bacillus, prostaglandin J2 (PGJ2) and laxative phenotypes showed the most significant associations. The above results suggest that ATTF may relieve constipation by regulating the gut microbiota and serotonergic synaptic pathway and has great potential for laxative drug development in the future.

Keywords: Amomum tsaoko; constipation; gastrointestinal motility; gut metabolites; gut microbiota.

Figure 1

The laxative active fraction of ATAE and its main chemical composition. Grouping and basic workflow for animal experiments 1 (A), 2 (C) and 3 (D). The defecation time of the first black stool (FBST) in animal experiments 1 (B), 2 (E), and 3 (F). Chemical composition of ATES (G) and ATTF (H) based on widely targeted metabolomics. The data are expressed as the means ± SEMs (n = 10–12). **, ***, compared with the CON group; #, ##, ###, compared with the LOP group. **, p < 0.01; ***, p < 0.001. #, p < 0.05; ##, p < 0.01; ###, p < 0.001.

Figure 2

Influence of ATTF on loperamide-induced constipation symptoms in mice. (A) Grouping and basic workflow of animal experiment 4. (B) The defecation time of the first black stool, FBST. (C) Wet weight of feces excreted in 6 h, FW. (D) Number of feces excreted in 6 h, FN. (E) Representative fecal morphology of each group. (F) Fecal water content. (G) Gastrointestinal transit rate, GTR. (H) Liver index. (I) Kidney index. (J) Ink advancing distance and intestinal length. (K) Photomicrographs of H&E-stained proximal colon sections. Infiltration of inflammatory cells into the tissue is shown in blue in the photomicrographs in the LOP group. The data are expressed as the means ± SEMs (n = 10–12). *, **, ***, compared with the CON group; #, ##, ###, compared with the LOP group. *, p < 0.05; **, p < 0.01; ***, p < 0.001. #, p < 0.05; ##, p < 0.01; ###, p < 0.001.

Figure 3

HAF restores the loperamide-induced gut microbial community structural and compositional shift. (A) The rarefaction curve of the Simpson index of each sample plateau at the ASV level. (B) Alpha diversity estimated by the Simpson index. (C) PCoA (principal coordinate analysis) plot based on binary Euclidean distance. (D) Linear discriminant analysis effect size (LEfSe) analyses (LDA score > 2.0). (E–K) Relative abundances of gut microbiota at the phylum, family, and genus levels, which were significantly affected by LOP or HAF, especially those reversed by HAF treatment. The data are expressed as the means ± SEMs (n = 6). *, compared with the CON group; #, ##, ###, compared with the LOP group. *, p < 0.05; #, p <0.05; ##, p < 0.01; ###, p < 0.001.

Figure 4

HAF changes the composition of loperamide-induced gut metabolites. Differential metabolites in the Venn diagram based on (A) cation detection and (B) anion detection. (C) Volcano map based on the LOP and CON groups. (D) Volcano map based on the LOP and HAF groups. (E) PCoA (principal coordinate analysis) based on all identified metabolites. (F) KEGG enrichment analysis. (G,H) Relative abundances of gut metabolites at the serotonergic synapse and arachidonic acid metabolism, respectively, which were significantly affected by loperamide or ATTF. (I) Relative abundances of butyric acid. The data are expressed as the means ± SEMs (n = 6). *, compared with the CON group; #, ###, compared with the LOP group. *, p < 0.05; #, p < 0.05; ###, p < 0.001. Log2FC, multiple change value of the expression difference of metabolites between the two groups; −Log10(p value), statistical test value of the difference in the expression of metabolites. The higher the value, the more significant the expression difference. p < 0.05, n = 8 in each group.

Figure 5

HAF increased the expression of gastrointestinal motility-related factors in the colon of STC mice. (A,B) Induced nitric oxide synthases (iNOS) and serotonin (5-HT) in serum. (C–G) The mRNA expression of serotonin 2 receptor (5-HT_2A), phospholipase A2 (PLA₂), cyclooxygenase-2 (COX2), transient receptor potential cation channel, subfamily A, member 1 (TRPA1), and Myosin light chain 3(MLC3) in the colon. The data are expressed as the means ± SEMs (n = 8). *, compared with the CON group; #, ##, ###, compared with the LOP group. *, p < 0.05. #, p < 0.05; ##, p < 0.01; ###, p < 0.001.

Figure 6

Heatmaps showing bivariate correlations between specific gut microbiota, gut metabolites and core host parameters in STC mice. (A) Correlations between the differential microbe taxa and differential host parameters. (B) Correlations between differential intestinal metabolites in serotonergic synapses and differential host parameters. (C) Correlations between the differential microbe taxa and differential intestinal metabolites in serotonergic synapses. The color at each intersection indicates the value of the r coefficient; p values were adjusted for multiple testing according to the BH procedures. * indicates a significant correlation between these two parameters (p < 0.05, n = 6 in each group).