Promotion of intestinal peristalsis by Bifidobacterium spp. capable of hydrolysing sennosides in mice

Abstract

Background: While there are a variety of identifiable causes of constipation, even idiopathic constipation has different possible mechanisms. Sennosides, the main laxative constituents of Daio, an ancient Kampo medicine, are prodrugs that are converted to an active principle, rheinanthrone, by intestinal microbiota. In this study, we aimed to determine the sennoside hydrolysis ability of lactic acid bacterial strains and bifidobacteria in the intestine and to investigate their effect on intestinal peristalsis in mice.

Methodology/principal findings: A total of 88 lactic acid bacterial strains and 47 bifidobacterial strains were evaluated for their ability to hydrolyze sennosides. Our results revealed that 4 strains, all belonging to the genus Bifidobacterium, had strong sennoside hydrolysis ability, exhibiting a decrease of >70% of sennoside content. By thin-layer chromatography analysis, rheinanthrone was detected in the medium cultured with B. pseudocatenulatum LKM10070 and B. animalis subsp. lactis LKM512. The fecal sennoside contents significantly (P<0.001) decreased upon oral administration of these strains as compared with the control. Intestinal peristalsis activity was measured by the moved distance of the charcoal powder administered orally. The distance travelled by the charcoal powder in LKM512-treated mice was significantly longer than that of control (P<0.05). Intestinal microbiota were analysed by real-time PCR and terminal-restriction fragment length polymorphism. The diversity of the intestinal microbiota was reduced by kanamycin treatment and the diversity was not recovered by LKM512 treatment.

Conclusion/significance: We demonstrated that intestinal peristalsis was promoted by rheinanthrone produced by hydrolysis of sennoside by strain LKM512 and LKM10070.

Figure 1. Time schedule of animal assay.

These tests were performed individually (one mouse/cage). (A) Sennoside-hydrolysing test by Bifidobacterium spp. Feces were collected every 2 hours. (B) Intestinal peristalsis promotion test by LKM512. This test was performed using a pair of mice: 1 LKM512-administered mouse and 1 control mouse.

Figure 2. Ability of lactic acid bacterial strains and bifidobacterial strains to hydrolyse Sennoside A and B.

Sennoside-hydrolysing values were expressed as the ratio of sennoside content in the medium without bacterial strain to that in the medium with bacterial strain.

Figure 3. Detection of rheinanthrone in sennoside-containing medium cultivated by bifidobacterial strain LKM10070 or LKM512 by thin-layer chromatography (TLC).

Spots of rheinanthrone were coloured yellow, and the Rf value was 0.68.

Figure 4. Sennoside hydrolysis in the intestine by bifidobacterial strain LKM10070 (A) and LKM512 (B) administered orally after kanamycin treatment.

Feces were collected every 2 hours up to 8 hours. The total sennoside contents in feces discharged up to 8 hours after administration of sennoside were significantly higher in kanamycin-treated mice than in the blank (no kanamycin) (p<0.001). These tests for the blank group were performed before kanamycin treatment. The amount of sennoside collected from the feces of mice administered LKM10070 (n = 8) or LKM512 (n = 9) was significantly lower than that collected from the feces of control mice (p<0.001, Mann-Whitney U test). Bars indicate the average value. The times of dosages are shown in Materials and Methods section and in Figure 1.

Figure 5. Effects of B. animalis subsp. lactis LKM512 on intestinal peristalsis of mice administered sennoside after kanamycin treatment.

(A) Picture of the intestinal tract. The activity of intestinal peristalsis was expressed as the distance travelled by the charcoal powder from the end of the ileum (i.e. the base of the caecum or start of the colon) to the top of the charcoal powder, measured using a ruler. (B) Comparison of the length of charcoal powder from the end of the ileum. This value was significantly longer in LKM512-adminstered mice (n = 5) than in the control (n = 5) (p<0.05), modified Wilcoxon signed-rank test). Error bars show the standard error of the mean.

Figure 6. Effects of kanamycin and LKM512 administration after kanamycin treatment on intestinal microbiota.

(A) Change in the number of total bacteria and B. animalis subsp. lactis LKM512 in feces. Since the detection limit of the standard curve is 10³ cells, bacterial counts below this limit were assigned a value of 10³ cells/g of feces. (B) Change in the T-RFLP profile after kanamycin treatment (middle) and LKM512 administration after kanamycin treatment (lower) on digestion with HaeIII. Arrow indicates T-RF predicted to be derived from LKM512.