Apigenin Impacts the Growth of the Gut Microbiota and Alters the Gene Expression of Enterococcus

Abstract

Apigenin is a major dietary flavonoid with many bioactivities, widely distributed in plants. Apigenin reaches the colon region intact and interacts there with the human gut microbiota, however there is little research on how apigenin affects the gut bacteria. This study investigated the effect of pure apigenin on human gut bacteria, at both the single strain and community levels. The effect of apigenin on the single gut bacteria strains Bacteroides galacturonicus, Bifidobacterium catenulatum, Lactobacillus rhamnosus GG, and Enterococcus caccae, was examined by measuring their anaerobic growth profiles. The effect of apigenin on a gut microbiota community was studied by culturing a fecal inoculum under in vitro conditions simulating the human ascending colon. 16S rRNA gene sequencing and GC-MS analysis quantified changes in the community structure. Single molecule RNA sequencing was used to reveal the response of Enterococcus caccae to apigenin. Enterococcus caccae was effectively inhibited by apigenin when cultured alone, however, the genus Enterococcus was enhanced when tested in a community setting. Single molecule RNA sequencing found that Enterococcus caccae responded to apigenin by up-regulating genes involved in DNA repair, stress response, cell wall synthesis, and protein folding. Taken together, these results demonstrate that apigenin affects both the growth and gene expression of Enterococcus caccae.

Keywords: Enterococcus; apigenin; gut microbiota; single molecule RNA sequencing.

Figure 1

The chemical structure of apigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one).

Figure 2

The effect of apigenin on the growth of single gut bacterial strains. Bacteria were inoculated in strain-specific media containing apigenin. McFarland unit values were determined using a densitometer at 0, 4, 8, 12, and 24 h post inoculation. The dotted line represents the control group in which bacteria grew with no apigenin added. The * mark indicates at least one experimental group was statistically significant from the control at that time point (p < 0.05). The 24 h growth curve with increasing concentrations of apigenin for (A) Lactobacillus rhamnosus GG; (B) Bacteroides galacturonicus; (C) Enterococcus caccae; (D) Bifidobacterium catenulatum.

Figure 3

Culture density and alpha diversity over time. The culture density for the control and apigenin (100 µg/mL) treated groups based on the OD₆₀₀ reading over time is represented using the left axis. The alpha diversity based on the Shannon index is represented using the right axis. The * mark indicates at least one experimental group was statistically significant from the control at that time point (p < 0.05).

Figure 4

Gut bacterial community composition over 48 h. Community composition was determined based on relative abundance. OTUs that were more than 0.1% in at least one time point are presented. Diversity of the community increased over time for both the control and apigenin (100 µg/mL) treated cultures.

Figure 5

Phylum composition of the gut microbiota community over time. Bioreactors containing basal media were inoculated with human fecal homogenate, and samples harvested at 4, 8, 12, 24, 48 h post inoculation. Microbial composition of each sample was determined by 16S rRNA sequencing, and percentages of the four major phyla for both the control and apigenin-treated groups were compared at each time point. The * mark indicates the experimental group was statistically significant from the control at that time point (p < 0.05). (A) Firmicutes; (B) Bacteroidetes; (C) Proteobacteria; (D) Actinobacteria.

Figure 6

Apigenin enhances growth of order Lactobacillus and genus Enterococcus in a community setting. The percent relative abundance for order Lactobacillus and genus Enterococcus was determined based on 16S rRNA sequencing. The * mark indicates that the experimental group (100 µg/mL apigenin) was statistically significant from the control at that time point, according to a 2-tailed, Student’s t-test (p < 0.05). (A) Lactobacillales order; (B) Enterococcus genus.

Figure 7

Major short chain fatty acid production by the gut microbial community over time. mmol/L is millimolar per liter. Amounts of the short chain fatty acids acetate, propionate, and butyrate were measured using a GC/MS. The * mark indicates the experimental group was statistically significant from the control at that time point (p < 0.05). (A) Acetate; (B) Propionate; (C) Butyrate.