Medications or dietary components can affect both the host and the host’s gut microbiota. Changes in the microbiota may influence medication efficacy and interactions. Daikenchuto (TU-100), a herbal medication, comprised of ginger, ginseng, and Japanese pepper, is widely used in Japanese traditional Kampo medicine for intestinal motility and postoperative paralytic ileus. We previously showed in mice that consumption of TU-100 for 4 weeks changed the gut microbiota and increased bioavailability of bacterial ginsenoside metabolites. Since TU-100 is prescribed in humans for months to years, we examined the time- and sex-dependent effects of TU-100 on mouse gut microbiota. Oral administration of 1.5% TU-100 for 24 weeks caused more pronounced changes in gut microbiota in female than in male mice. Changes in both sexes largely reverted to baseline upon TU-100 withdrawal. Effects were time and dose dependent. The microbial profiles reverted to baseline within 4 weeks after withdrawal of 0.75% TU-100 but were sustained after withdrawal of 3% TU-100. In summary, dietary TU-100 changed mouse microbiota in a time-, sex-, and dose-dependent manner. These findings may be taken into consideration when determining optimizing dose for conditions of human health and disease with the consideration of differences in composition and response of the human intestinal microbiota.
The gut microbiota has a systemic effect on human health [
Daikenchuto (TU-100) is a herbal medication used under the aegis of the traditional Kampo therapeutic system in Japan. Kampo has been used for over a thousand years and TU-100 is widely used in Japan to improve gastrointestinal (GI) motility, especially in the lower GI tract, including prevention of postoperative paralytic ileus. TU-100 is an aqueous extract from a mixture of 50% ginger
Although it was approved in 1986 by the Japanese government as a pharmaceutical compound and various mechanisms to explain the effect of TU-100 on improving GI tract motility and blood flow have been proposed [
Since TU-100 can be prescribed long term in clinical settings, to understand its mechanisms of action, it is important to delineate TU-100’s effects in terms of long-term exposure, dose dependency, sex specificity, and reversibility of the effects after cessation of TU-100 exposure in an in vivo model. Therefore, we undertook a long-term, 24-week study, to investigate the chronological change of the gut microbiota in the presence and absence of TU-100 administration, its dose dependence on TU-100, and its reversibility after withdrawal of TU-100 in female and male mice. We believe that this is the first comprehensive study to examine the effect of a herbal medication on the gut microbiota.
C57Bl/6J mice were bred in-house at the University of Chicago Animal Care Facilities (Institutional Animal Care and Use Committee Protocol 71084). Five breeding pairs were set up when mice were 7–9 weeks old and called the F0 generation. Their progeny were termed the F1 generation and were used to set up 25 breeding pairs, with care being taken to avoid using littermates for any breeding pair. For the present study, mice of the F2 generation were used. The F2 generation pups were weaned at 21 days after birth and ear tagged for identification. Fresh bedding is provided every 14 days in our animal facility. Between weaning and the start of the experiments (see below), to decrease cage-cage variability, a mixed bedding protocol was implemented. Thus, 3-4 days and also at 8–10 days after fresh bedding was provided, the mouse bedding was removed and mixed. The bedding from all cages of females and male animals was mixed together and the mixed bedding was distributed back to all cages. Mice were maintained on a 12-hour light 12-hour dark cycle, with light initiated at 6 AM.
Mice were fed Teklad Global 18% Protein Rodent Diet (2018) (Envigo, Madison, WI, USA) until one week before the start of experiments and then switched to the defined, AIN-76A Purified Diet (CA. 170481) (The American Institute of Nutrition, 1977) (Envigo) for one week. TU-100 was obtained as a powder from Tsumura & Co. (Ami, Ibaraki, Japan). TU-100 was included at 0.75, 1.5, or 3% wt/wt in AIN76A (Institutional Animal Care and Use Committee protocol 72101). The dosage of TU-100 for murine experiments was based on previous reports that aimed to achieve blood concentrations of major TU-100 compounds, similar to that reported in human data [
Forty-eight female and 48 male mice were used for the first experiment and were 7–12 weeks old at the start of the experimental protocol, labeled as “week 0.” Mice were assigned to 3 groups (groups 1–3) and each group contained 16 female and 16 male mice. Mice in group 1 and group 2 were fed AIN-76A and 1.5% TU-100 for 24 weeks (weeks 0–24), respectively. Group 3 was fed 1.5% TU-100 for 12 weeks (weeks 0–12) and then returned to AIN-76A for another 12 weeks (weeks 12–24). Half of the mice in each group were sacrificed at week 12 and the remaining mice at week 24 (Figure
Fecal pellets were harvested from the mice every 4 weeks from week 0 through week 24 (Figure
DNA was extracted from stool pellets by standard, published protocols [
Mann–Whitney
Analysis of fecal microbiota was performed before switching mice to a defined diet to determine the variability of the starting population. 16S rRNA genes were sequenced and principal coordinate analysis (PCoA) plots generated by QIIME from MiSeq data for 48 female and 48 male samples. Since 6 samples from female mice gave less than 5000 sequences, these samples were excluded. The PCoA plots showed the similar distribution between sexes in both unweighted and weighted UniFrac distances (Figure
Mice were placed into 3 groups (groups 1–3) in each sex of littermates. As shown in Figure
To determine how quickly the microbiota changes after addition to the diet and how quickly the changes are reversible, samples were analyzed from fecal DNA at weeks 0, 4, 8, 12, 16, 20, and 24 and the data are shown in Figure
16S rRNA sequences were next analyzed by QIIME for taxonomy assignment. We analyzed the composition of phyla in each group at weeks 0, 12, and 24 of female and male samples (Figure
Phyla with more than 1.0% of population at week 24 with/without 1.5% TU-100.
Phylum | Group 1 | Group 2 | Group 3 | |
---|---|---|---|---|
(%) | (%) | (%) | (group 1 versus group 2) | |
Female samples | ||||
Bacteroidetes | 28.95 ± 6.66 | 49.13 ± 10.13 | 42.02 ± 14.77 | 0.0012 |
Deferribacteres | 3.30 ± 1.55 | 3.08 ± 5.94 | 0.67 ± 0.68 | ns |
Firmicutes | 46.23 ± 6.91 | 37.37 ± 10.49 | 45.78 ± 14.46 | ns |
Proteobacteria | 20.13 ± 3.67 | 8.89 ± 3.02 | 10.64 ± 2.51 | 0.0003 |
| ||||
Male samples | ||||
Actinobacteria | 0.15 ± 0.05 | 1.74 ± 1.40 | 1.46 ± 2.33 | 0.0006 |
Bacteroidetes | 40.39 ± 7.89 | 36.53 ± 11.15 | 38.03 ± 9.11 | ns |
Deferribacteres | 2.23 ± 2.87 | 3.43 ± 4.27 | 1.62 ± 1.75 | ns |
Firmicutes | 40.99 ± 10.44 | 41.92 ± 3.64 | 39.95 ± 7.48 | ns |
Proteobacteria | 14.77 ± 6.19 | 14.97 ± 9.10 | 17.73 ± 8.29 | ns |
Genera with more than 1.0% of population showing significant alterations by 1.5% TU-100 administration for 24 weeks.
Female samples
Genus level | Group 1 | Group 2 | Group 3 | |
---|---|---|---|---|
(%) | (%) | (%) | (group 1 versus group 2) | |
k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Bacteroidaceae;g__Bacteroides | 0.85 ± 0.43 | 3.53 ± 1.33 | 2.45 ± 1.71 | 0.0012 |
k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__ | 1.46 ± 0.89 | 4.66 ± 1.87 | 1.44 ± 1.06 | 0.0012 |
k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__S24-7;g__ | 14.12 ± 2.51 | 24.93 ± 5.93 | 20.13 ± 7.24 | 0.0093 |
k__Bacteria;p__Firmicutes;c__Bacilli;o__Turicibacterales;f__Turicibacteraceae;g__Turicibacter | 0.16 ± 0.29 | 1.50 ± 1.37 | 0.92 ± 1.24 | 0.0022 |
k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__;g__ | 23.34 ± 5.09 | 14.45 ± 4.46 | 21.38 ± 13.40 | 0.0059 |
k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Clostridiaceae;g__ | 0.18 ± 0.07 | 1.13 ± 0.64 | 0.62 ± 0.47 | 0.0003 |
k__Bacteria;p__Proteobacteria;c__Deltaproteobacteria;o__Desulfovibrionales;f__Desulfovibrionaceae;g__ | 17.05 ± 4.75 | 7.35 ± 2.69 | 8.85 ± 2.19 | 0.0003 |
|
Male samples
Genus level | Group 1 | Group 2 | Group 3 | |
---|---|---|---|---|
(%) | (%) | (%) | (group 1 versus group 2) | |
k__Bacteria;p__Actinobacteria;c__Coriobacteriia;o__Coriobacteriales;f__Coriobacteriaceae;g__ | 0.03 ± 0.02 | 1.18 ± 1.25 | 0.77 ± 1.48 | 0.0006 |
k__Bacteria;p__Firmicutes;c__Bacilli;o__Turicibacterales;f__Turicibacteraceae;g__Turicibacter | 1.82 ± 1.88 | 0.05 ± 0.08 | 0.17 ± 0.17 | 0.0200 |
k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__[Ruminococcus] | 1.47 ± 0.45 | 0.75 ± 0.19 | 0.85 ± 0.30 | 0.0006 |
k__Bacteria;p__Firmicutes;c__Erysipelotrichi;o__Erysipelotrichales;f__Erysipelotrichaceae;g__Allobaculum | 5.33 ± 3.52 | 17.71 ± 6.29 | 15.44 ± 6.84 | 0.0006 |
|
To determine if the effects of dietary TU-100 are dose dependent, diets were prepared with half and twice the TU-100 (0.75 and 3% wt/wt) as used in experiment 1. For this experiment, 5 groups of male mice were used as shown in Figure
We next analyzed the chronological change of fecal microbiota in each group using fecal DNA samples at weeks 0, 4, 8, 12, 16, 20, and 24 (Figure
We examined the gut microbial composition in each group at weeks 0, 12, and 24 (Figure
Phyla with more than 1.0% of population at week 24 with/without 0.75%/3.0% TU-100.
Phylum | Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | | |
---|---|---|---|---|---|---|---|
(%) | (%) | (%) | (%) | (%) | (group 1 versus group 3) | (group 1 versus group 5) | |
Actinobacteria | 1.17 ± 0.64 | 0.67 ± 0.57 | 0.31 ± 0.13 | 0.31 ± 0.29 | 0.59 ± 0.53 | 0.0317 | ns |
Bacteroidetes | 41.58 ± 8.45 | 39.99 ± 9.61 | 44.80 ± 6.26 | 33.43 ± 9.79 | 45.84 ± 19.47 | ns | ns |
Deferribacteres | 1.17 ± 1.11 | 1.97 ± 3.31 | 3.67 ± 4.05 | 5.01 ± 4.56 | 6.15 ± 7.07 | ns | ns |
Firmicutes | 42.05 ± 3.17 | 42.67 ± 3.72 | 38.41 ± 10.00 | 39.58 ± 7.33 | 34.20 ± 7.70 | ns | ns |
Proteobacteria | 13.33 ± 6.69 | 14.11 ± 5.40 | 11.8 ± 2.06 | 20.87 ± 11.30 | 12.58 ± 8.96 | ns | ns |
Genera with more than 1.0% of population showing significant alteration by 0.75%/3.0% TU-100 administration for 24 weeks.
Genus level | Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | | |
---|---|---|---|---|---|---|---|
(%) | (%) | (%) | (%) | (%) | (group 1 versus group 3) | (group 1 versus group 5) | |
k__Bacteria;p__Actinobacteria;c__Coriobacteriia;o__Coriobacteriales;f__Coriobacteriaceae;g__ | 1.03 ± 0.56 | 0.60 ± 0.54 | 0.08 ± 0.04 | 0.21 ± 0.26 | 0.06 ± 0.04 | 0.0159 | 0.0159 |
k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__ | 1.19 ± 0.41 | 0.91 ± 0.72 | 5.43 ± 2.70 | 3.36 ± 0.80 | 3.66 ± 1.95 | 0.0159 | 0.0317 |
k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__AF12 | 0.79 ± 0.33 | 0.74 ± 0.28 | 1.41 ± 0.32 | 1.77 ± 0.92 | 1.34 ± 0.15 | ns | 0.0317 |
k__Bacteria;p__Bacteroidetes;c__Bacteroidia;o__Bacteroidales;f__Rikenellaceae;g__Rikenella | 0.42 ± 0.33 | 0.32 ± 0.22 | 1.30 ± 0.27 | 1.42 ± 0.64 | 0.56 ± 0.33 | 0.0159 | ns |
k__Bacteria;p__Firmicutes;c__Bacilli;o__Lactobacillales;f__Streptococcaceae;g__Lactococcus | 0.59 ± 0.26 | 0.71 ± 0.55 | 1.44 ± 0.38 | 1.29 ± 1.31 | 0.84 ± 0.70 | 0.0159 | ns |
k__Bacteria;p__Firmicutes;c__Clostridia;o__Clostridiales;f__Lachnospiraceae;g__[Ruminococcus] | 1.05 ± 0.42 | 0.67 ± 0.20 | 0.90 ± 0.64 | 0.87 ± 0.23 | 0.54 ± 0.17 | ns | 0.0317 |
|
The present study demonstrates the effect of TU-100 administration and withdrawal on fecal microbiota in both female and male mice and the effects of different doses in male mice for 24 weeks. Our study provides insight into determining the clinical efficacy and safety of TU-100 and its influence on the microbiota by examining the following parameters: (1) both female and male animals were studied, (2) chronological changes over a long (24 weeks) observation period, (3) analyses of reversibility of microbiota after cessation of TU-100, and (4) analyses of the dose-dependent effects of TU-100. It is well known that the intestinal microbiota in mice differs from humans. TU-100 may alter the intestinal microbiota by providing substrates for certain bacterial species, increasing their abundance, or some components may provide compounds with antimicrobial actions against certain intestinal bacteria. It is not possible to predict what changes might occur in the human intestinal microbiota from changes in phyla and genera that changed in mice. However, if the bacteria have similar properties in the human and mouse microbiota, changes may occur and remained to be determined.
A general consideration of microbiota studies is to minimize variations in the starting microbiota between groups prior to experimentation, that is, to start with a consistent baseline. It is increasingly recognized that, even within the same facility, and using the same mouse strain, there are room to room and cage to cage differences that influence the microbial profile. For this purpose, in the present study, we adopted a protocol of mixed bedding among all cages in each experiment from weaning until the start of the experiments as described in the Materials and Methods. The plots in each group appeared to be randomly distributed in PCoAs at the start point among each sex and experiment. We attribute this to our methodology of bedding transfers. The mixed bedding protocol is thought to reduce the variability of fecal microbiota among cages safely (Miyoshi et al., manuscript in preparation).
In the present study, the doses of TU-100 were determined to achieve blood concentrations of major TU-100 compounds similar to those in human [
Our recent report using the same murine model demonstrated that dietary TU-100 modulates the transcript and protein expression of drug metabolizing enzymes and drug transporters in the liver, small intestine, and colon in a dose- and sex-dependent manners and that in most cases the effects were reversible after cessation of TU-100 treatment [
Our analysis of the changes of phyla and genera revealed interesting differences between sexes. Bolnick et al. [
We therefore addressed the question of whether there was variability between our experiments 1 and 2. In experiment 2, the genus of the family of Rikenellaceae increased with both 0.75% and 3.0% TU-100 and the genus of the family Coriobacteriaceae decreased with both 0.75% and 3.0% TU-100, and some genera showing significant change with one dose of TU-100 presented the same trend of change with the other dose. Taken together, the alterations of the microbiota after 0.75% and 3.0% TU-100 administration for 24 weeks appeared to be similar. On the other hand, the changes of genera with 1.5% TU-100 seemed different from the changes with 0.75% and 3.0% TU-100 (Tables
We also examined the change of 16S rRNA OTUs, recognizing that these data cannot resolve beyond a genus level and provide no functional information. Among females and males in experiment 1, many OTUs that belong to the family of Desulfovibrionaceae decreased and many that belong to the genus
The administration of dietary TU-100 alters murine fecal microbiota over time for 24 weeks. These changes are time, sex, and dose dependent. At higher doses of TU-100, the changes in gut microbiota are more rapid, pronounced, and sustained. Whether TU-100 changes human intestinal microbiota will determine if similar changes occur.
Gastrointestinal tract
Quantitative Insights into Microbial Ecology
Operational taxonomic units
Principal coordinate analysis.
The authors declare that they have no conflicts of interest regarding the publication of this paper.
This work was supported by a grant from Tsumura & Co. (EBC), NIH grants Digestive Disease Center grant P30 DK42086 (EBC), NIH RO1 DK097268 (EBC), and support from the GI Research Foundation of Chicago (EBC). The authors thank Mrinalini Rao for her help in editing the manuscript.
Supplementary Figure 1: the difference in male fecal microbiotas at week 0 between experiments 1 and 2. (A) The PCoA plots of unweighted and weighted UniFrac distances were generated for male fecal microbiotas at week 0 in experiments 1 (red squares) and 2 (blue circles). Both PCoA plots showed the different distribution between experiments. (B) The composition of phyla at week 0 of male samples in both experiments is assigned. Supplementary Table 1: the OTUs with significant increase and decrease by 1.5% TU-100 administration for 24 weeks. Supplementary Table 2: the OTUs with significant increase and decrease by 0.75% TU-100 administration for 24 weeks. Supplementary Table 3: the OTUs with significant increase and decrease by 3.0% TU-100 administration for 24 weeks.