Efficacy of Glycicumarin and Isoliquiritigenin in Suppressing Colonic Peristalsis in Both an Animal Model and a Clinical Trial

Abstract

Patients with diarrhea-predominant irritable bowel syndrome (IBS-D) show excessive peristalsis, and antispasmodic agents may be useful therapeutic agents. There are few reports on the use of Kampo medicines for the treatment of IBS-D. Shakuyakukanzoto (SKT) is a Kampo medicine that is effective against abdominal pain. We examined the relationship between SKT and intestinal peristalsis in an animal model and a prospective study. In the animal model, SKT and its components were administered from the serosal side of the colon and colonic peristalsis was evaluated using intraluminal pressure and spatiotemporal mapping before and after the administration of SKT and its components. In this clinical trial, we used abdominal ultrasonography (US) to obtain long-axis images of the sigmoid colon of 11 patients. The frequency of intestinal peristalsis was measured using US in five patients with SKT and six patients without medication after the ingestion of a test meal. The primary outcome was the frequency of peristalsis. The Clinical Trial Registry Website (Trial No. UMIN-CTR; UMIN000051547). In the animal model, peony did not suppress peristalsis frequency, but SKT (p = 0.005) and glycyrrhiza (p = 0.001) significantly suppressed peristalsis frequency compared with saline and peony. Among the glycyrrhiza components, glycycoumarin and isoliquiritigenin suppressed the peristalsis frequency compared to dimethyl sulfoxide (control) (p = 0.001, 0.01, respectively). In a clinical trial, peristalsis was significantly suppressed after oral administration in patients taking SKT (p = 0.03). Administration of SKT was found to inhibit colonic peristalsis, with glycicumarin and isoliquiritigenin being particularly relevant among its components.

INTRODUCTION

According to the Functional Gastrointestinal Disorders Guideline 2020, irritable bowel syndrome (IBS) has four potential classifications.¹⁾ In diarrhea-predominant IBS (IBS-D), which is one of the classifications, increased intestinal peristalsis has been reported.²⁾ Therefore, intestinal peristalsis inhibitors are used in the treatment of IBS-D.¹⁾ Hyoscine butylbromide and other drugs are sometimes used to suppress peristalsis, but their use is limited owing to adverse events and insufficient effectiveness.^1,3,4)

The use of Kampo medicines for the treatment of IBS has also been mentioned, and one Kampo medicine, keishikashakuyakuto, has been reported to significantly suppress abdominal pain in patients with IBS-D compared to placebo.⁵⁾ However, evidence of the utility of Kampo medicine is limited. The components of keishikashakuyakuto include peony and glycyrrhiza, which have anti-spasmodic effects.^6,7) Shakuyakukanzoto (SKT) is a Kampo medicine composed of peony and glycyrrhiza, and is often used to treat muscle cramps. It is occasionally used to treat abdominal pain, and its mechanism is reported to be derived from the inhibition of smooth muscle of the intestinal tract.⁶⁾ In addition, SKT reportedly suppresses peristaltic motion and has antispasmodic effects during gastrointestinal endoscopy and barium enema.^8,9)

Peony and glycyrrhiza themselves are each composed of several components. The plasma concentrations of active components after SKT revealed glycyrrhetic acid (GA), glycyrrhetic acid-3-O-monoglucuronide (GAG), glycycoumarin (GCM), and isoliquiritigenin (ILG) in glycyrrhiza, paeoniflorin (PAE), and albiflorin (ALB) in peony.¹⁰⁾ SKT has been reported to promote intestinal smooth muscle relaxation via significant anticholinergic and phosphodiesterase 3 inhibitory actions.^11,12) However, the mechanisms underlying SKT-induced smooth muscle inhibition remain unclear.

We hypothesized that the administration of one of the components of SKT to the colon would result in less peristalsis by relaxing the intestinal smooth muscle. Therefore, we examined the effects of SKT on smooth muscle and the mechanisms involved in animal models and clinical trials.

MATERIALS AND METHODS

Animals

Sprague–Dawley rats (7–12 weeks old) were obtained from Shimizu Laboratory Sciences Co., Ltd. (Kyoto, Japan). The animals were housed at 22 °C under a 12-h d/night cycle and allowed access to food and ad libitum access to water. Male rats were used in the experiments.

The Measurement of Intraluminal Pressure of Isolated Rats Proximal Colon Tract

Rats were fasted overnight, euthanized by decapitation, and their colons were removed. Experiments were conducted in special settings based on previous reports.¹³⁾ In brief, we placed a 2- to 3-cm segment of the proximal colon in an organ bath (100 mL volume) and perfused the bath with Krebs solution (34–35 °C, 3.5 mL/min). We securely attached the oral and anal ends of the proximal colon segment to the saline input and output ports, respectively. To monitor intraluminal pressure (cmH₂O), we set a Mikro-Tip catheter pressure transducer (SPR-524; Millar Instruments, Houston, TX, U.S.A.) in the colon. Intestinal peristalsis was induced by increasing the drain tube by loading an intraluminal pressure of approximately 4 cmH₂O. Intraluminal pressure waves were evaluated by using a data acquisition and analysis system (MP100; BIOPAC Systems, Goleta, CA, U.S.A.). Motility was macroscopically observed by spatiotemporal mapping using video images (HDC-HS 100-K; Panasonic, Osaka, Japan).

Experimental Protocol and Chemicals and Drugs

In experiments of the proximal colon, after recording 20–25 min of motor activity, drugs were added to the organ bath, and recordings were continued for a further 20–25 min. Information on where to purchase chemicals and how to use them is provided below: SKT, peony, and glycyrrhiza powders were purchased from Tsumura & Co. (Tokyo, Japan). SKT was dissolved in saline solution (1 mL). SKT was dissolved in saline solution (1 mL) at 10.1, 25.25, 50.5, 101, and 202 mg, respectively, and the effect of each concentration on intestinal peristalsis was examined. Peony and glycyrrhiza were dissolved in a saline solution (1 mL). We added them to Krebs solution (100 mL) on the serosal side of the colon in the organ bath, finally, the concentrations of SKT were 0.1, 0.25, 0.5, 1.0 , and 2.0 mg/mL. GA, GAG, GCM, ILG, PAE, and ALB were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), Nagara Science Co., Ltd. (Gifu, Japan), and Sigma-Aldrich Chemical Co. (St. Louis, MO, U.S.A.), respectively.

The concentrations of GA, GAG, GCM, ILG, PAE, and ALB were determined based on the following reference after SKT determining the concentration.^14–16) GA, GGA, GCM, and ILG were dissolved in dimethyl sulfoxide (DMSO) (1 mL), and PAE and ALB were dissolved in saline solution (1 mL) and then added to Krebs solution (100 mL) on the serosal side of the colon in an organ bath. After the experiments, using the measured intraluminal pressure waves and spatiotemporal mapping, we calculated the peak frequency (PF) as the frequency of high-amplitude pressure peaks (>8 mm on spatiotemporal mapping) per minute within a certain period. We also calculated the area under the curve (AUC) above the minimum pressure value during a defined period. We calculated the peak pressure amplitude (PPA) as the average pressure of the peaks minus the minimum value during a defined period. We calculated PPA, PF, and AUC for each period as the ratio before drug administration.

Spatiotemporal Mapping

Based on previous reports, we created spatiotemporal mapping using image-based representations of motion.^17,18) We sampled a still image of the colon every 5 s from the video. Using ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.), we calculated the colon width (coded as image intensity, black to white) at each point along the length of the colon (image y-axis) for each video frame (image x-axis). Broad relaxation of the intestinal tract is represented by the white area. Strong intestinal contractions are indicated by the red diagonal stripes.

Study Design and Patients

This is a prospective study. The subjects of this study were patients with abdominal discomfort or those taking SKT, a gastrointestinal peristalsis inhibitor, who underwent abdominal ultrasonography (US) for screening at our institution.

Patients were excluded if they had 1) obvious infection or inflammation; 2) serious complications (intestinal paralysis, intestinal obstruction, interstitial pneumonia or pulmonary fibrosis, diabetes mellitus that is difficult to control, renal failure, or liver cirrhosis); 3) inflammatory bowel disease or postoperative esophagus, stomach, duodenum, or large intestine; and 4) age <20 years. Written informed consent was obtained from all the patients to participate in this study.

Abdominal US was performed in 15 consecutive patients with abdominal discomfort or use of antispasmodic drugs in July 2023. Four patients declined to participate in this study. Ultimately, 11 patients were enrolled in this study and provided a written informed consent. Five patients were taking SKT for abdominal comfort, and the other six did not take any medication.

Procedures

There are various methods of evaluating the function of colonic motility, such as an examination using radiopaque markers to measure the transit time through the digestive tract, and in addition, methods of evaluating colonic motility using abdominal US and cine magnetic resonance imaging (MRI) as imaging tests have also been reported.^19–25) Based on previous reports, we evaluated motility patterns in the sigmoid colon using US.¹⁹⁾ The sigmoid colon was confirmed from the body surface by US, and a US probe was placed in the longitudinal direction of the sigmoid colon to delineate a long-axis image of the sigmoid colon centered at the site where it crosses the iliopsoas muscle. We used the APLIO i800 TUS-AI800 ultrasonic diagnostic instrument (Canon Medical Systems, Tokyo, Japan), Aplio i700 TUS-AI700 ultrasonic diagnostic instrument (Canon Medical Systems), and Arietta 850 US system (FUJIFILM Healthcare, Tokyo, Japan). The probes were 3.5-MHz convex or 7.5-MHz linear probes.

As shown in Fig. 1, the protocol was to observe the peristalsis of the sigmoid colon for 20 min while the patient had been fasting for 9–15 h after ingesting a normal diet and then to use 200 mL (400 kcal) of TERMEAL 2.0α^® (TERMEAL; Terumo Corporation, Tokyo, Japan) as a liquid test meal, followed by resting for 10–20 min. Peristalsis was then observed for 20 min after the ingestion of the test meal. Subsequently, SKT (2.5 g), purchased from Tsumura & Co. (Tokyo, Japan), and water (100 mL) or water only (100 mL) was administered orally, and peristalsis was observed for 20 min. Peristalsis of the sigmoid colon was evaluated in terms of the contraction pattern and frequency. The evaluation included changes in intestinal peristalsis during fasting, intake of TERMEAL, and the internal administration of SKT/water. As previously reported, the frequency of segmental contractions without peripheral propagation or propulsion and those that propagated peripherally were evaluated, and their frequency were measured.¹⁹⁾ The ratio of change in peristalsis 10 min before and after SKT/water administration was calculated. Two experts performed the US examination.

Fig. 1. Patient Flow Chart

Eleven patients underwent abdominal ultrasonography after fasting for 9–15 h after ingesting a normal meal.

All procedures were video recorded, and the frequency of peristalsis was measured using video movies after the examination. The videos were evaluated by three physicians and the frequency of peristalsis was measured as the average of these evaluations.

Statement of Ethics

All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyoto Prefectural University of Medicine (Kyoto, Japan) under Assurance Numbers M2022-144, M2022-146, and M2022-47 and were performed in accordance with the institutional guidelines for the care and use of laboratory animals, which is in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

This clinical study was approved by the Ethics Committee of Kyoto Prefectural University of Medicine (ERB-C-2733). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and the 1964 Declaration of Helsinki and its later versions. This study followed the CONSORT guidelines and was registered with the University Hospital Medical Network Clinical Trials Registry (UMIN000051547). Informed consent was obtained from all patients for inclusion in the study.

Statistical Analyses

The median, mean, range, and 95% confidence intervals were summarized in the quantitative data. We compared patient characteristics and details of the colonoscopy examinations using the chi-square test, chi-square test with Yates’ correction, unpaired t-test, and Mann–Whitney U test. Using the chi-square test, we compared the proportion of patients with no peristalsis after administration. We compared the changes in the peristalsis scores of the patients. After drug administration, the Wilcoxon signed-rank test was performed. We evaluated the statistical significance between two groups using an unpaired t-test and among three groups using one-way ANOVA or Kruskal–Wallis test. All analyses were performed using GraphPad Prism 7 software (GraphPad Software, San Diego, CA, U.S.A.) on a Windows-based computer.

RESULTS

Evaluating Rat Colonic Motor Activity by Video Imaging and Intraluminal Pressure Peaks Along with the Effect of SKT and Its Components

Using video image recording and intraluminal pressure measurements, we analyzed the activity of isolated segments of the rat proximal colon. High-amplitude pressure peaks were observed at the beginning of the experiments. Administration of saline to the serosal side of the colon had no effect on the intraluminal pressure or spatiotemporal mapping (Fig. 2a).

Fig. 2. Change in Colonic Motor Activity and Intraluminal High-Amplitude Pressure in Rats

(a) The colonic motility and intraluminal high-amplitude pressure after administration of room-temperature saline. (b) A typical pattern of reduction in colonic motility and intraluminal high-amplitude pressure after shakuyakukanzoto (SKT) administration. (c) Serial photographs of colonic motor activity. Left: motility before SKT administration. Right: motility after SKT administration.

Regarding concentration, a dose of 50.5 mg of SKT (0.5 mg/mL) or higher was found to inhibit peristalsis (Supplementary Fig. 1). In this study, 101 mg of SKT was dissolved in 1 mL of saline and added to Krebs (1.0 mg/mL). Peony and glycyrrhiza were 50.5 mg, respectively (0.5 mg/mL). GA was 0.5 mg, GGA was 0.5 mg, and GCM was 1 mg, ILG was 0.06 mg, PAE was 5 mg ALB was 1 mg, respectively. Administration of SKT, glycyrrhiza, GCM and ILG on the serosal side of the colon induced low-amplitude periodic pressure peaks (Figs. 2b, 3a, 4a, b). Serial photographs of the mobility of the unmedicated colon (left) and the colon treated with SKT (right) are shown in Fig. 2c. After the administration of SKT, the spastic colon started to relax (Supplementary Video 1). Figure 3 shows the suppression of the pressure peak pattern, PF, AUC, and PPA, and the typical pattern of reduction in colonic motility and intraluminal high-amplitude pressure after glycyrrhiza administration. The suppression of PF in the groups treated with SKT and glycyrrhiza was significantly lower than that in the saline and peony groups (p = 0.005, 0.001, one-way ANOVA) (Fig. 3b). The suppression of the AUC in the group treated with glycyrrhiza was significantly lower than that in the peony group (p = 0.03, one-way ANOVA) (Fig. 3c). Administration of saline, SKT, peony, or glycyrrhiza had no significant effect on the suppression of PPA (one-way ANOVA) (Fig. 3d).

Fig. 3. Changes in Colonic Motor Activity and Intraluminal High-Amplitude Pressure by SKT and Peony and Glycyrrhiza in Rats

Suppression of peak frequency (PF) after the administration of saline (1 mL), shakuyakukanzoto (SKT) (101 mg), peony (50.5 mg), and glycyrrhiza (50.5 mg) in rats. (a) Typical pattern of reduction in colonic motility and intraluminal high-amplitude pressure after glycyrrhiza administration. The pattern of colonic motor activity was similar to that with SKT. High-amplitude pressure peaks on the intraluminal pressure chart disappeared temporarily after glycyrrhiza administration but were observed again after a short time. In spatiotemporal mapping, the entire area was white, reflecting dilation of the colon due to peristaltic inhibition. (b) Decreased ratio of PF under high-amplitude pressure following administration of SKT and glycyrrhiza. To quantify the decrease in high-amplitude pressure induced by SKT and glycyrrhiza, the ratio of contraction frequency before and after administration with a length of >8 mm was calculated as the %PF. ** p < 0.01 compared to saline, peony. (c) Area under the curve (AUC) at high-amplitude pressure. The AUC was calculated before and after drug administration, using the lowest intraluminal pressure value as the baseline. The suppression of AUC in the group treated with glycyrrhiza was significantly lower than that in the peony groups. * p < 0.05 compared to peony. (d) The peak pressure amplitude (PPA) at high-amplitude pressure. The difference between the highest and lowest intraluminal pressure was defined as the PPA, and the ratio of the PPA before and after medication was calculated as the % PPA. There were no significant differences among the four groups (n = 5). When the high-amplitude pressure peaks appeared, the red line became visible.

The pattern of colonic motor activity in the glycyrrhiza was similar to that in the SKT (Fig. 3a). High-amplitude pressure peaks on the intraluminal pressure chart disappeared after glycyrrhiza administration; however, after a short time, high-amplitude pressure peaks were observed again. Spatiotemporal mapping revealed that the entire area was white, reflecting dilation of the colon due to peristaltic inhibition. After the administration of glycyrrhiza, the spastic colon started to relax (Supplementary Video 2).

When the high-amplitude pressure peaks appeared, the red line became visible. In Fig. 4, the suppression of the pressure peak pattern, PF, AUC, and PPA, and the typical pattern of reduction in colonic motility and intraluminal high-amplitude pressure after GCM and ILG administration are also shown. The suppression of PF in the GCM group was significantly lower than that in the DMSO, GA, and GAG groups (p = 0.001, one-way ANOVA) (Fig. 4c). The suppression of PF in the ILG group was significantly lower than that in the DMSO and GAG groups (p = 0.04, one-way ANOVA) (Fig. 4c). The suppression of PF in the ILG group tended to be lower than that in the GA group (p = 0.11, one-way ANOVA) (Fig. 4c). The administration of DMSO, GCM, ILG, GA, or GAG had no significant effect on the suppression of PPA or AUC (one-way ANOVA) (Figs. 4d, e). The pattern of colonic motor activity in the GCM and ILG was similar to that in the SKT and glycyrrhiza (Figs. 4a, b). High-amplitude pressure peaks on the intraluminal pressure chart disappeared temporarily after glycyrrhiza administration but were observed again after a short time. Spatiotemporal mapping revealed that the entire area was white, reflecting dilation of the colon due to peristaltic inhibition. After the administration of GCM and ILG, the spastic colon started to relax (Supplementary Videos 3, 4). In Supplementary Fig. 2, the PF and PPA AUC after the administration of saline, PAE, and ALB are also shown. The administration of saline, PAE, and ALB had no significant effect on the suppression of PF, PPA, or AUC (one-way ANOVA) (Supplementary Figs. 2a–c).

Fig. 4. Changes in Colonic Motor Activity and Intraluminal High-Amplitude Pressure by the Active Components of SKT and in Rats

Suppression of peak frequency (PF) after administration of dimethylsulfoxide (DMSO) (1 mL), glycyrrhetic acid (GA) (0.5 mg), glycyrrhetic acid-3-O-monoglucuronide (GAG) (0.5 mg), glycycoumarin (GCM) (1 mg) and isoliquiritigenin (ILG) (0.06 mg) in rats. (a) Typical pattern of reduction in colonic motility and intraluminal high-amplitude pressure after GCM administration. The pattern of colonic motor activity was similar to those of SKT and glycyrrhiza. High-amplitude pressure peaks on the intraluminal pressure chart disappeared temporarily after glycyrrhiza administration but were observed again after a short time. In spatiotemporal mapping, the entire area was white, reflecting dilation of the colon due to peristaltic inhibition. When the high-amplitude pressure peaks appeared, the red line became visible. (b) Typical pattern of reduction in colonic motility and intraluminal high-amplitude pressure after ILG administration. The pattern of colonic motor activity was similar to that observed after the administration of SKT and glycyrrhiza. High-amplitude pressure peaks on the intraluminal pressure chart disappeared temporarily after glycyrrhiza administration but were observed again after a short time. In spatiotemporal mapping, when the high-amplitude pressure peaks appeared, the red line became visible. (c) The rate of PF was decreased under high-amplitude pressure after the administration of GCM and ILG. ** p < 0.01 vs. DMSO, GA, and GAG. * p < 0.05 vs. DMSO, and GAG. (d) AUC at high-amplitude pressure. The AUC was calculated before and after drug administration, using the lowest intraluminal pressure value as the baseline. There were no significant differences among the four groups. (e) Peak pressure amplitude (PPA) at high-amplitude pressure. The difference between the highest and lowest intraluminal pressure was defined as the PPA, and the ratio of the PPA before and after medication was calculated as the % PPA. There were no significant differences among the four groups (n = 3).

Changes in Intestinal Peristalsis by Internal SKT on US

Peristalsis was evaluated in 11 patients. Five patients took SKT orally for abdominal discomfort, and the other six patients did not take any antispasmodic agents. The clinical characteristics of the patients are summarized in Table 1. No significant differences were found in the characteristics of the two groups. Evaluation of intestinal peristalsis using US was possible in all cases. After the test meal, the frequency of peristalsis increased, the intestinal lumen widened, and the muscle layer thickened.

Table 1. Patient Characteristics

	Water	SKT	p-Value
Number of patients	6	5
Age, mean ± S.D. (range)	40.0 ± 7.9 (31–54)	47.4 ± 9.0 (33–60)	0.85
Sex, % (n) male/female	100.0/0.0 (6/0)	100.0/0.0 (5/0)	—
Drinking % (n) Habitual/occasional/none	16.7/33.3/50.0 (1/2/3)	40.0/20.0/40.0 (2/1/2)	0.68
History, % (n)
Heart disease	0.0 (0)	0.0 (0)	—
Diabetes	0.0 (0)	0.0 (0)	—
Cerebral infarction	0.0 (0)	0.0 (0)	—
Abdominal disease	0.0 (0)	0.0 (0)	—
Inflammatory bowel disease	0.0 (0)	0.0 (0)	—
Irritable bowel syndrome	0.0 (0)	0.0 (0)	—
History of abdominal surgery	16.7 (1)	0.0 (0)	—
Medicine, % (n)
Peristalsis inhibitors	0.0 (0)	0.0 (0)	—
Laxatives	0.0 (0)	0.0 (0)	—
Stimulant laxatives	0.0 (0)	0.0 (0)	—
Other laxatives	0.0 (0)	0.0 (0)	—
Probiotics	0.0 (0)	0.0 (0)	—
Antidepressants	0.0 (0)	0.0 (0)	—
Characteristics of stools % (n) Diarrhea/constipation/mixed/normal	0.0/16.7/16.7/66.6 (0/1/1/4)	40.0/0.0/0.0/60.0 (2/0/0/3)	0.25

SKT, shakuyakukanzoto; S.D., standard deviation.

In the water group, the frequency and strength of peristalsis further increased. However, in the SKT group, the lumen of the intestinal tract became narrower, the muscle layer became thinner, and the frequency of peristalsis decreased (Figs. 5a–c) (Supplementary Video 5). In the SKT group, who had been taking SKT, intestinal peristalsis decreased to 44.9% after the administration of SKT, while in the water group, who had not been taking SKT, intestinal peristalsis increased to 125.7% after the administration of water; intestinal peristalsis was significantly reduced in the SKT group. (p = 0.03, unpaired t-test) (Fig. 5d).

Fig. 5. Changes in the Colonic Motor Activity on Abdominal Ultrasonography before and after Internal Administration of Shakuyakukanzoto (SKT)/Water

(a) Abdominal ultrasonography (US) of the sigmoid colon 1 min before the internal administration of SKT. Approximately 20 min had passed since the ingestion of TERMEAL, and the intestinal tract had a spastic form under the influence of the occurrence of intestinal peristalsis. Thickening of the muscle layer was also observed. (b) Visual 5 min after the internal administration of water. The muscle layer remained as thick as before water ingestion, and the lumen remained dilated. (c) Visual 5 min after the internal administration of SKT. The muscle layer was thinner and the lumen narrower than before SKT administration. (d) A decreased ratio of intestinal peristalsis was observed before and after the internal administration of SKT. * p < 0.05 compared to water.

Adverse Events

We observed no symptoms after examinations in any of the patients.

DISCUSSION

In this study, we examined the effects of SKT on colonic motility using intraluminal pressure, video imaging of the proximal colon of rats, and a clinical trial. The results of the animal model experiments suggest that administration of SKT, especially GCM and ILG, may be associated with a significant decrease in colonic peristalsis. The results of the clinical trial showed that the oral administration of SKT significantly suppressed peristalsis in the human colon.

SKT is a Kampo medicine with peony and glycyrrhiza as ingredients. It has been reported to have antispasmodic and analgesic effects on skeletal and smooth muscles, and is used in daily clinical practice to relieve cramps and pain in the extremities, abdomen, and trachea etc.⁶⁾ It was reported that SKT dissolved in 50 mL of saline solution, and when sprayed directly from the endoscope during colonoscopy, it significantly suppressed colonic motility 3 min after spraying.⁸⁾ Furthermore, SKT dissolved in warm water and sprayed on the duodenum during endoscopic retrograde cholangiopancreatography (ERCP) suppressed peristalsis during examination.⁹⁾ However, the components of SKT that exert antispasmodic effects on colonic peristalsis remain unclear. Therefore, in the present study, we examined the function of each component of SKT in colonic peristalsis in a basic experiment.

In an animal model, SKT significantly reduced the frequency of colonic peristalsis in rats immediately after adding SKT. SKT is an extract powder composed of peony and glycyrrhiza combined at a ratio (g) of 1 : 1; therefore, this result suggests that glycyrrhiza and peony may either act individually or interact to contribute to the inhibition of intestinal peristalsis. It has been reported that peony inhibits the release of acetylcholine from cholinergic nerves, but not by suppressing the action of acetylcholine, and glycyrrhiza inhibits the action of acetylcholine on smooth muscles.⁶⁾ In the present study, we showed that glycyrrhiza, but not peony, had an inhibitory effect on peristalsis. Therefore, we hypothesized that glycyrrhiza was involved in the inhibition of peristalsis and focused on its constituents. We examined the components of glycyrrhiza that affect peristalsis. We also conducted experiments on the peony components: PAE and ALB. However, they did not inhibit the intestinal peristalsis. Our animal model differed from that of Maeda et al. This could be the reason why peony did not inhibit peristalsis in the present study. It was reported that when SKT is administered as a single oral dose of 2.5 g or 5.0 g/d per person, GCM, GA, and GAG were identified in the plasma as the active components of SKT. GCM, GA, and GAG are absorbed into the human blood, with GCM and ILG reaching their maximum blood concentration at 15 min.¹⁰⁾ This report suggests that the blood concentrations of GCM and ILG increased early after oral administration. In the present study, GA, GAG, GCM, and ILG were administered to the colon of rats, and significant inhibition of intestinal contractions was observed with GCM and ILG, with peristalsis promptly suppressed after the addition of GCM and ILG. Thus, GCM and ILG may contribute to peristalsis suppression.

GCM is a major bioactive coumarin isolated from glycyrrhiza.²⁶⁾ It is rapidly absorbed into the blood when administered orally, and unchanged GCM is found to be the main form in both plasma and urine.²⁷⁾ Factors associated with intestinal peristalsis include smooth muscle cells, interstitial cells of Cajal (ICCs), and the enteric nervous system (ENS).^28–32) Regarding the relationship between GCM and smooth muscle cells, GCM has been reported that GCM acted as a potent antispasmodic through the intracellular accumulation of cAMP via the inhibition of phosphodiesterases (PDEs), especially isozymes 3.¹²⁾ Increased cAMP activates protein kinase, phosphorylates myosin light chain kinase, and reduces the ability of calcium ions to bind to calmodulin, ultimately relaxing smooth muscle cells.²⁵⁾ ICCs are pacemaker cells in the gastrointestinal tract, and the ICC network is coupled to smooth muscle via gap junctions.²⁹⁾ During the depolarization phase of the pacemaker potential, current flows through the gap junctions and depolarizes the membrane potential of the smooth muscle until it reaches an action potential discharge threshold, inducing smooth muscle contraction. However, there have been no reports on the relationship between GCM and ICCs. Calcium ion concentrations in the endoplasmic reticulum reservoir may determine the frequency of pacemaker activity, and calcium ion concentrations may also be important for ICCs.³²⁾ The ENS is reported to be the largest neural organ outside the brain, including a full repertoire of sensory, interneurons, and motor neurons that collectively detect luminal contents, drive secretory function, and control intestinal motility.^30,31) ILG is a flavonoid isolated from glycyrrhiza.³³⁾ It has also been reported to inhibit intestinal contractions in the jejunum, ileum, and rectum, and the relaxant effect of ILG seems to have no relationship with its inhibition of PDEs.³⁴⁾ ILG has been reported to play a dual role in regulating gastrointestinal motility by activating muscarinic receptors and blocking calcium channels.³³⁾ GA and GAG are metabolites of glycyrrhizic acid (GL) present in Glycyrrhiza radix.^10,35) GA and GAG have been reported to be effective for skin and antiviral, antimicrobial, hepatoprotective, and anti-inflammatory activities.³⁶⁾ There are no reports of GA GAGs being associated with intestinal peristalsis, and the present results are consistent with this finding. GCM is a representative coumarin isolated from Glycyrrhiza. ILG is a flavonoid with a chalcone structure. Both have natural dimeric forms, and their natural dimeric forms could be associated with the suppression of intestinal peristalsis.^37,38)

The results of this basic experiment suggested that SKT is involved in the suppression of intestinal peristalsis. Therefore, we designed our clinical study to investigate colonic peristalsis in patients receiving SKT for abdominal discomfort following oral administration. Abdominal ultrasonography was performed during a clinical trial. Various methods have been used to assess colonic motility.^19–25) We chose to use abdominal US because it is easier and less burdensome for the patient and allows us to check for colonic peristalsis in real time. The results of this clinical study suggest that SKT inhibits the peristaltic frequency in the human colon. This was the first study to observe colonic peristalsis in real time before and after oral administration of SKT and showed a significant reduction in the frequency of colonic peristalsis 10 min after oral SKT administration. The limitations of this study were that abdominal US was performed in a small number of cases, and although the videos were reviewed by several people, the examinations were performed by two experts. In addition, we used abdominal US, which has poor reproducibility, and the usual colonic peristalsis might have differed between patients who received SKT and those who did not. The measurement of PPA and AUC was not precise because the intestinal tract dilated when the drug with antispasmodic effects was sprayed in the animal model, which caused the baseline intraluminal pressure to rise. SKT has been suggested to inhibit colonic peristalsis and be useful in suppressing colonic peristalsis in diseases that present with excessive peristalsis. Glycycoumarin and isoliquiritigenin, components of SKT, have been suggested to contribute to peristaltic inhibition.

Acknowledgments

We thank all the members of the Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Yuji Omiya, and Kunitsugu Kubota (Tsumura Research Laboratories, Tsumura & Co., Ibaraki, Japan).

Funding

This work was supported by the Japanese Foundation for Research and Promotion of Endoscopy Grant (JSPS KAKENHI Grant Nos. JP19K21243, JP22K20899) and the Medical Technology Research and Development Grant Project to Promote Kyoto-Origin Innovation.

Author Contributions

Reo Kobayashi: lead of data curation, formal analysis, project administration, and writing of original draft; Ken Inoue: lead of conceptualization, funding acquisition, investigation, methodology, project administration, resources, software, supervision, and visualization, data curation, formal analysis, and support for writing of original draft; Satoshi Sugino: support of formal analysis and validation; Ryohei Hirose: support of data curation and methodology; Toshifumi Doi, Akihito Harusato, Osamu Dohi, Naohisa Yoshida, Kazuhiko Uchiyama, Takeshi Ishikawa, and Hideyuki Konishi: support of data curation; Tomohisa Takagi: methodology and project administration; Yasuko Hirai: lead of formal analysis and visualization; Katsura Mizushima: lead of data curation and formal analysis, and support of methodology; Yuji Naito: Funding acquisition: Lead; Software: Lead; Supervision: Lead; Yoshito Itoh: Supervision: Lead; Writing—review & editing: Lead.

All authors had access to the study data and reviewed and approved the final manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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