Rhein 8-O-β-D-Glucopyranoside Elicited the Purgative Action of Daiokanzoto (Da-Huang-Gan-Cao-Tang), Despite Dysbiosis by Ampicillin

Abstract

Sennoside A (SA), the main purgative constituent of Daiokanzoto (da-huang-gan-cao-tang; DKT), is generally regarded as a prodrug that is transformed into an active metabolite by β-glucosidase derived from Bifidobacterium spp. It has been suggested that antibiotics would promote dysbiosis, and thereby inhibit the purgative activity of DKT. In this study, ampicillin was administered to mice for 8 d, and the changes in the SA metabolism of SA alone and of DKT were investigated. The results showed that the SA metabolism of SA singly continued to be inhibited by ampicillin, but that of DKT was activated from day 3 under the same conditions. In order to investigate the mechanism of SA metabolism activated by DKT in the mice administered ampicillin, changes in the SA metabolism were observed in the presence of rhein 8-O-β-D-glucopyranoside (RG) in rhubarb and liquiritin in glycyrrhiza, both of which accelerated the SA metabolism. In fact, RG achieved an activation of SA metabolism similar to that by DKT. The purgative action of DKT, which was continued treatment of the ampicillin, was significantly greater than that by SA alone, and it was shown that RG was involved in this effect. We also analyzed changes in the intestinal microbiota before and after administration of ampicillin. No Bifidobacteria were detected throughout the treatment, but the population of Bacteroides was significantly increased after 3 d under the same conditions. Taken together, these results strongly suggested that the RG in DKT changed the function of Bacteroides and thereby allowed DKT to metabolize SA.

Kampo is a system of traditional Japanese medicine developed from traditional medicine practices in ancient China. Kampo medicines incorporate multiple herbal medicines, they exhibit various pharmacological actions and are prescribed for a range of diseases. Daiokanzoto (da-huang-gan-cao-tang; DKT) is a Kampo medicine that consists of rhubarb and glycyrrhiza; it was demonstrated to be useful for constipation in a clinical double-blind study.^1,2) The purgative action of DKT is generally ascribed to the transformation of sennoside A (SA), the main laxative constituent in rhubarb, to an active metabolite, rheinanthrone, by the β-glucosidase derived from Bifidobacterium spp.^3–5) (Fig. 1). The purgative action of the SA conversion to rheinanthrone has been explained in terms of Na⁺/K⁺ transport, water and mucus secretion in the colon.^6,7) This process, which also involves aquaporin-3, was described in detail in recent report.⁸⁾ We have also demonstrated that the metabolic activity of SA in intestinal microbiota was significantly accelerated when rhein 8-O-β-D-glucopyranoside (RG) in rhubarb or liquiritin (LQ) in glycyrrhiza coexisted with SA.^9–11) Moreover, the purgative action of SA was significantly intensified when RG and LQ were co-administered orally to mice.^10,12) These results show that the influence of these constituents on the fate of SA-derived rheinanthrone may promote the purgative action of SA. Taken together, these studies provide evidence of an effective interaction between DKT, a Kampo medicine with multiple constituents.

Fig. 1. Metabolic Pathway of Sennoside A by Intestinal Microbiota

As of 2015 in Japan, 294 Kampo formulas had been approved as over the counter (OTC) Kampo formulations, and 148 Kampo formulations were being prescribed clinically. However, these medicines are usually prescribed concomitantly with Western medicines, which poses a risk of interaction.^13–17) Kampo medicines contain many kinds of glycosides as the main and/or active constituents. Most glycosides tend to be metabolized by enzyme-producing intestinal microbiota before being absorbed into the body. Therefore, the administration of Kampo formulations in combination with medicines promoting dysbiosis, such as antibiotics, can lead to problematic interactions.^13,14) So far, we revealed that the purgative action of SA and DKT was inhibited by several antibiotics to mice in a single injection.¹²⁾ However, a preliminary experiment suggested that the metabolism of SA under continuous administration of β-lactam antibiotics differed according to whether DKT or SA alone was added.

In this study, we identified a constituent of DKT that plays a role in the difference in metabolic and purgative actions between DKT and SA during continuous ampicillin administration in mice. Furthermore, we confirmed that this constituent brings about changes in the ampicillin-induced distribution of intestinal microbiota. Our findings shed light on the efficacy of DKT, a Kampo medicine with multiple constituents, and suggest its proper usage.

MATERIALS AND METHODS

Materials

The herbal medicine pieces for decoction in DKT, rhubarb (kinmon-daio in Japanese, Lot No. 100901) and glycyrrhiza (touhoku-kanzo in Japanese, Lot No. 050202), were purchased from Tochimototenkaido (Osaka, Japan). Sennoside A and ampicillin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). RG was isolated from rhubarb and was identified according to the method in a previous paper.¹⁰⁾ Liquiritin was isolated from glycyrrhiza, and its structure was identified by comparing its ¹H- and ¹³C-NMR data with those reported in the literature.¹⁸⁾ Ultrapure distilled water was prepared with deionized-distilled water. All other chemicals were analytical reagent- or HPLC-grade commercial products.

Preparation of Freeze-Dried Extract

The freeze-dried extract of DKT (4 g of rhubarb and 1 g of glycyrrhiza; yield: 2.25±0.04 g; sennoside A: 14.0±0.13 mg/g; RG: 93.4±2.69 mg/g; liquiritin: 61.2±1.82 mg/g extract) was prepared as follows: chopped herbal drugs were boiled with 500 mL of water on an electric heater for 40 min, filtered, and lyophilized.

Animal Preparation

Animal experiments were all carried out in accordance with the Guidelines for Animal Experimentation of Fukuyama University. Male ddY mice, weighing 30–40 g, were obtained from SHIMIZU Laboratory Supplies (Kyoto, Japan) and housed in a 12 h light–dark cycle at 21 to 24°C for at least one week before the experiments. They were given free access to food and water before the start of experiments.

Continuous Administration of Ampicillin

Ampicillin at 25 mg/kg, three times the human daily dose, was mixed with the powder chow and administered to mice for 8 d.

Chromatographic Conditions

The HPLC apparatus was an Agilent Technologies 1100 Series system (Waldbronn, Germany) consisting of a binary pump, an autosampler, a thermostatted column compartment, and a photodiode array (PDA) detector. All modules and data collection were controlled by Agilent Chemstation software. The column was a TSKgel ODS-80TsQA reversed-phase column, with a packing-particle size of 5 µm, 150×4.6 mm i.d. (Tosoh, Tokyo, Japan). The mobile phase was a gradient system with 200 µL·L⁻¹ (ca. 0.017%) phosphoric acid in water (A) and acetonitrile (B), which were deaerated by sonication before use. The elution program was performed from 19 to 20% B for 3 min, 20 to 21% B for 5 min, a stepwise increase to 43% B for 8 min, and a stepwise increase to 90% B for 5 min for washing. A re-equilibration period of 12 min was used between individual runs. The flow rate was 1.0 mL·min⁻¹. The injection volume was 5 µL. The column temperature was 50°C. The detection wavelength was set at 265 nm for determination and in the range of 200 to 500 nm for validation of peak purity.

Assaying the Metabolic Activity of SA by Mouse Intestinal Microbiota

Fresh feces obtained from mice were homogenized in 5 volumes of 0.01 M potassium phosphate buffer (pH 7.4) by bubbling with CO₂ gas to eliminate air. The fecal suspension was centrifuged at 1500×g for 1 min, and the supernatant was used as an intestinal microbiota mixture. DKT (20 mg/mL) was prepared with 0.01 M potassium phosphate buffer and 0.5% sodium hydrogen carbonate. SA (0.4 mM) was prepared with 0.01 M potassium phosphate buffer. RG and LQ, each at a concentration of 2 mM, were prepared with 0.5% sodium hydrogen carbonate. The assay samples were prepared by mixing equal amounts of SA and each constituent sample. Tubes containing the assay samples (0.25 mL) and the fecal suspension (1 mL) were incubated at 37°C for 4 h under anaerobic conditions. Anaerobic procedures were carried using an anaerobic jar with an AnaeroPack (Mitsubishi Gas Chemical, Tokyo, Japan). The reaction was immediately stopped by adding 0.425% (v/v) phosphoric acid in methanol (1.25 mL). After centrifugation at 1500×g for 5 min, the supernatant was passed through a Minisart RC 15 (Japan Sartorius, Tokyo, Japan) and subjected to HPLC. The blank process involved an incubation mixture at 0 min, as mentioned above. Metabolic rate was calculated by the percentage of the content of SA in the incubation mixture compared with that in the blank.

The Purgative Action of SA, Daiokanzoto and SA+RG

Mice were isolated in a wire-bottomed cage covered with a beaker (11×15 cm), which was placed on blotting paper. The condition of feces was observed 1 h before administration of each sample, and only the mice that excreted normal feces were used. SA (80 mg/kg) was prepared with 0.01 M potassium phosphate buffer. RG at a dose of 44.6 mg/kg was prepared with 0.5% sodium hydrogen carbonate. The SA with RG sample was prepared by mixing equal amounts of SA and RG sample and orally administered to mice in a single dose. DKT (1.21 g/kg) was prepared with 0.01 M potassium phosphate buffer and 0.5% sodium hydrogen carbonate, and orally administered to mice in a single dose. We have confirmed that the concentration of DKT and SA was equally the purgative activity. After the oral administration of sample, the condition of feces was observed at intervals of 1 h for 8 h. The feces in the worst condition were graded into three consistency levels as follows: 0, normal; 1, soft; and 2, unformed.¹⁹⁾ The total feces score was the mean value for the total consistency level measure each hour in each mouse.

Analysis of Intestinal Microbiota by Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Fecal samples of mice were analyzed by targeting the bacterial 16S ribosomal RNA (rRNA) genes that were previously identified by T-RFLP by Nagashima et al.^20,21) The fluorescently labeled 516f primer (5′-TGC CAG CAG CCG CGG TA-3′; Escherichia coli positions 516–532) and 1510r primer (5′-GGT TAC CTT GTT ACG ACT T-3′; Escherichia coli positions 1510–1492) were used to amplify the 16S rRNA genes. The 5′-ends of the forward primers were labeled with 5′-carboxyfluorescein (5-FAM), which was synthesized by Life Technologies (Tokyo, Japan). The amplification products were subjected to gel electrophoresis in 2% agarose followed by ethidium bromide staining. The purified polymerase chain reaction (PCR) products were digested with the restriction enzyme of BslI (New England BioLabs, Beverly, MA, U.S.A.). The lengths of the fluorescently labeled terminal restriction fragments (T-RFs) were analyzed by electrophoresis on an automated sequence analyzer (ABI PRISM 3100-Avant Genetic Analyzer; Applied Biosystems) in GeneScan mode. DNA fragments from MapMarker 1000 (Bio Ventures Inc.) were used as standard size markers. The POP-7 polymer (Applied Biosystems) was used as a separation matrix. The lengths and peak areas of T-RFs were determined with Gene Mapper v5. software (Applied Biosystems).

Statistical Analysis

Data are shown as the mean±standard deviation (S.D.). Statistical comparisons between two groups were made by means of unpaired t-test or Mann–Whitney’s U test (KyPlot; Kyence Inc.). To compare more than two groups, a Steel’s test or Steel–Dwass test for multiple comparisons (KyPlot, Kyence Inc.) was used. A probability value of p<0.05 was considered to indicate statistical significance.

RESULTS

Metabolic Activity of SA and Daiokanzoto under Ampicillin Administration

In a preliminary test, we showed that the metabolism of SA differed according to whether DKT or SA alone was administered during continuous β-lactam antibiotic administration in mice. In this study, we administered β-lactam antibiotics to mice for 8 d to induce dysbiosis and investigated changes in the SA metabolism of DKT and SA alone. As shown in Fig. 2, SA metabolism of SA alone continued to be significantly inhibited for 1 d after ampicillin administration as compared with the control (non-ampicillin-administered) group. In contrast, that of DKT was inhibited for the first 2 d of ampicillin treatment, but it was activated after 3 d, and after 8 d there was no difference between the ampicillin-administered group and the control. These results suggested that a constituent of DKT induced the metabolism of SA by affecting the intestinal microbiota or their functions, despite dysbiosis by ampicillin. Therefore, the changes in the SA metabolism could be attributed to the presence of RG in rhubarb or LQ in glycyrrhiza, either or both of which could have accelerated the metabolic activity of SA. Therefore, we next examined the changes in the SA metabolism in the presence of RG and LQ. We found that the metabolism of SA was not significantly different between the addition of LQ and SA alone, and thus the ability of LQ to accelerate the metabolism of SA disappeared with ampicillin treatment. However, the SA metabolism by the addition of RG was inhibited until day 2 of ampicillin administration, but the SA metabolism was activated on day 3, and the level of SA metabolism was not different from that of the control group after day 4 of ampicillin administration. Taken together, these results showed that there was clear difference in the SA metabolism under ampicillin administration between DKT and SA alone. This difference was attributed to the RG in DKT changing the diversity of the intestinal microbiota, which induced dysbiosis by ampicillin, and/or their functions, so that RG activated the SA metabolism of DKT.

Fig. 2. The Activating Effect of Rhein 8-O-β-D-Glucopyranoside (RG) on the Metabolism of Sennoside A (SA) Is Inhibited by Ampicillin

(A) SA; (B) Daiokanzoto; (C) SA+Liquiritin; (D) SA+RG. Each point represents the mean±S.D. of 3 samples with (●) or without (○) ampicillin. Statistical significance (*** p<0.001) was examined by Student’s t-test.

The Purgative Activity of SA and DKT under Ampicillin Administration

Our results on the SA metabolism activated by DKT suggested that RG contributed to the metabolism of SA. In order to clarify whether RG is involved in the purgative action of SA under ampicillin administration, the feces scores were compared between mice administered DKT, those administered SA with RG and those administered SA alone. In addition to SA, various other constituents of rhubarb exhibit purgative activity, and RG could also have mild purgative activity. In a previous study, the purgative activities of RG (22.3–44.5 mg/kg) were observed over 10 h. The purgative score of RG was about 0.6 at a dose of 44.5 mg/kg, and purgative activity was not observed at a dose of 22.3 mg/kg. Therefore, the dose of RG was set at 22.3 mg/kg. This concentration of RG was approximately 1/4 at concentration of RG included in DKT (1.21 g/kg). As shown in Fig. 3, mice treated with ampicillin exhibited a significant decrease in purgative activity not only in the group administered SA alone but also in that administered DKT and SA with RG as compared with the control group. However, the purgative activity of DKT under ampicillin treatment was significantly higher than that in the mice administered SA alone, and it was proved that RG was significantly involved in these effects. Thus, it was demonstrated that RG was involved in the purgative action of DKT activated under the ampicillin administration.

Fig. 3. The Activating Effect of Rhein 8-O-β-D-Glucopyranoside (RG) on the Purgative Activity of Sennoside A (SA) Is Inhibited by Ampicillin

Each column represents the mean ±S.D. of 7 mice. ^† p<0.05, ^††† p<0.001, significant vs. the control by Mann–Whitney’s U test. ** p<0.01, significant difference from SA pre-administered with ampicillin by Steel’s test.

Analysis of Intestinal Microbiota before and after Ampicillin Administration by Means of T-RFLP

The activating effect of DKT on SA metabolism under ampicillin treatment demonstrated that RG activated the metabolism of SA. Thus, it was to clarify that RG altered the intestinal microbiota or their functions, and we confirmed changes in the diversity of intestinal microbiota before and after the ampicillin administration by means of a T-RFLP. This study employed the methods of Nagashima and confirmed the changes in bacterial species reported by that author.^20,21) As shown in Fig. 4, Bifidobacterium accounted for approximately 20% of the total microbiota before the ampicillin administration, but was not detected at all after the ampicillin treatment. On the other hand, Bacteroides were not detected at all after the ampicillin treatment on day 1, but they were significantly increased after 3 d of ampicillin treatment. The other bacterial species were not significantly changed between before and after the ampicillin treatment. Thus, it was strongly suggested that Bacteroides are not able to have the ability of the SA metabolism unless RG alters their functions.

Fig. 4. Analysis of Intestinal Microbiota Treated with Ampicillin

The letters correspond to the following phylogenetic bacterial groups: (A) Bifidobacterium; (B) Bacteroides; (C) Clostridium cluster IV; (D) Clostridium cluster IX; (E) Clostridium subcluster XIVa; (F) Others. Statistical significance (* p<0.05) was examined by Steel–Dwass test (n=10).

DISCUSSION

Kampo medicines are orally administered, and they contain many kinds of glycosides as the main and/or active constituents. Most glycosides may be metabolized by the intestinal microbiota, which produce a wide range of enzymes. Thus understanding the relation between the intestinal microbiota and the metabolism of orally administered glycosides is highly important for understanding the pharmacological effects.^22–24) DKT is clinically effective for constipation.^1,2) The purgative action of DKT is generally ascribed to the transformation of SA, the main laxative constituent in rhubarb, to an active metabolite, rheinanthrone, by the β-glucosidase derived from Bifidobacterium.^4,5) We confirmed the contribution of Bifidobacterium to the SA metabolism in our previous report, and it was assumed that the RG in rhubarb intensified the activation of SA by increasing the synthesis of SA-metabolic enzyme derived from Bifidobacterium.¹¹⁾ In this study, the SA metabolism of SA alone continued to be inhibited under the ampicillin treatment. In contrast, it was elucidated that of DKT was activated under ampicillin treatment. At the same time, we observed changes in the diversity of intestinal microbiota by means of T-RFLP, and Bifidobacterium was not detected at all throughout the ampicillin treatment period. However, the levels of Bacteroides were significantly increased along with the recovery of SA metabolism of DKT and SA with RG. Bacteroides develops natural resistance to antibiotics, and particularly to penicillin antibiotics, by producing β-lactamase, and several reports have shown that the ratios of Bacteroides are increased in the presence of β-lactams, such as ampicillin.^25–27) Our present finding that Bacteroides were increased by continuous ampicillin treatment tended to be similar to these previous reports. From the results on purgative action, which continued throughout the eight days of ampicillin treatment, the purgative action of SA was inhibited, but that of DKT was increased, which proved that RG was involved in these effects. However, the purgative action of DKT and SA with RG groups was significantly decreased as compared with that in the non-treated ampicillin group. This was considered to be due to the annihilation of Bifidobacterium by the ampicillin treatment. It was assumed that Bacteroides did not have the ability to metabolize SA by themselves, unless the RG in DKT altered their functions such as β-glucosidases and/or reductases. Based on these findings, we searched for the β-glucosidases of Bacteroides species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Most of the β-glucosidases of Bacteroides have been classified into the Glycoside Hydrolase family 3. Therefore, it was strongly suggested that the RG in DKT conferred the ability to metabolize SA onto not only Bifidobacterium but also Bacteroides by changing their functions. Enzymes produced by intestinal microbiota are essential proteins for microbiota in which cannot lack to grow themselves, because it reserves their energy and the intestinal environment for survival and metabolizes substances threatening against themselves. Many of the constituents inhibiting the function of these enzymes are known, very few of the constituents activating their functions have been reported. Thus, to demonstrate in detail the mechanism of action of RG is pivotal to understanding the function of intestinal microbiota such as Bifidobacterium and Bacteroides. To demonstrate the mechanism of action of RG, which accelerating effect of SA metabolism, we are attempting to use the β-glucosidases of Bacteroides to clone genes and express proteins. We plan to confirm the accelerating effect of RG on the SA metabolism after assessing the kinetic parameters in carbohydrate metabolism and SA metabolism for these β-glucosidases.

If patients with constipation are prescribed antibiotics, particularly penicillin-group antibiotics, these agents should been chosen not so much sennosides preparation as DKT positively. In this study, we shed light on the usefulness of DKT as an agent based on multiple constituents, versus Western medicines with a single constituent.

In recent years, genome-based approaches have allowed us to provide a perspective on human-microbiota mutualism and disease.²⁸⁾ The high bacterial richness and diversity in intestinal microbiota may be viewed as a positive attribute. In contrast, changes and imbalances in intestinal microbiota, due to biased food intake and antibiotics administration, are associated with altered health states. Many reports have suggested relationships between intestinal microbiota and diseases, such as allergic inflammation,^29–31) metabolic diseases,^32,33) and obesity,^34,35) and central nervous system diseases.^36–38) We considered that these findings may be relevant to our present results. The characteristic efficacy of Kampo medicine is predicted to involve not only the metabolism of the constituents of Kampo medicines by intestinal microbiota but also changes in the diversity and/or functions of intestinal microbiota caused by the Kampo constituents. Namely, the characteristic efficacy of Kampo medicines is a result of the crosstalk between their constituents and the intestinal microbiota. We designed to demonstrate a working hypothesis of the above-mentioned. Kampo medicines are prescribed for various diseases due to their specific pharmacological action. However, it is not enough to scientific elucidation of Kampo medicine, which elicited the characteristic efficacy. Taking this study as a trigger, we have undertaken a detailed characterization of the crosstalk between the constituents of Kampo medicines and intestinal microbiota.

Acknowledgments

The authors are grateful to Ms. Naoka Sakamoto, Ms. Ami Sugino, Mr. Tsutomu Ogura, Ms. Azusa Kobayashi, Ms. Erika Shinchi and Ms. Shiori Morishita for their technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

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