Acanthopanax senticosus Root Extract Exerts Dual Action on Mouse Ileal Smooth Muscle Function, Leading to Modulation of Gastrointestinal Motility

Abstract

Diarrhea is often caused by changes in lifestyle, stress, or side effects of drugs. Acanthopanax senticosus root extract (ASRE) has long been used as a functional food remedy with anti-fatigue, neuroprotective, and immunomodulatory activities. However, it is unclear whether ASRE has beneficial effects on gastrointestinal (GI) motility. Therefore, we first investigated whether ASRE directly affects contractile functions of the isolated mouse ileum, and then assessed its effects on GI transit of a charcoal meal in normal mice and a carbachol (CCh)-induced diarrhea mouse model. ASRE caused contraction of the isolated mouse ileum and the maximum contraction was approximately half of that induced by acetylcholine (ACh) administration. In the presence of atropine, this ASRE-induced contraction disappeared, while relaxation responses were observed. However, ASRE reduced potassium chloride- and ACh-induced contractions, and the inhibitory effect was not counteracted by a β-blocker. Administration of a nitric oxide synthase inhibitor or potassium channel blockers did not affect the ASRE-induced relaxation. Oral administration of ASRE for 1 and 4 d reduced the increased GI transit in CCh-treated but did not affect the GI transit of normal mice. These results indicate that ASRE exhibited dual effects of contraction via muscarinic receptors and direct relaxation on mouse ileal function, and its relaxant effect could be useful in treating diarrhea symptoms, resulting in an increase in the parasympathetic nerve activities.

INTRODUCTION

Gastrointestinal (GI) disorders, such as constipation and diarrhea, often occur because of factors such as lifestyle disturbances, stress, and side effects of drugs, and such unpleasant symptoms adversely affect the QOL. Irritable bowel syndrome, one of the most common functional bowel disorders,¹⁾ is characterized by recurrent abdominal pain, discomfort, and bloating. Physical or emotional stress or both are considered important factors that exacerbate symptoms of this syndrome.^1–3) Dysfunction of the autonomic nervous system is considered one of the factors contributing to the pathogenesis of this syndrome.⁴⁾ However, the etiology is complex and appears to be multi-factorial.²⁾ Laxative agents are widely used for the treatment of GI motility disorders, including irritable bowel syndrome, but they have undesirable side effects such as nausea, abdominal pain, constipation, and diarrhea.^5–7)

Many supplements commonly used against GI disorders were developed from folk/herbal remedies with traditional applications, for example, peppermint oil, artichoke, and turmeric.⁶⁾ Acanthopanax senticosus Harms, family Araliaceae (AS, previously classified as Eleutherococcus senticosus), which is commonly known as Siberian ginseng or Ciwujia, is used as a traditional Chinese medicine preparation in China, Korea, Russia, and Japan.^8,9) AS is used to treat stress-induced physical and mental changes^8,10) because it has anti-fatigue,^10–12) neuroprotective,^13–16) and immunomodulatory effects.^17–22) According to the Chinese Pharmacopoeia, the dried root and rhizome or stem of AS are widely prescribed to nourish the qi, fortify the spleen, tonify the kidney, and tranquilize the mind.⁸⁾ AS root extract (ASRE) has been reported to have anxiolytic effects by regulating autonomic nervous system functions.²³⁾ This neural modification raises the possibility that ASRE could improve GI disorders such as constipation, which commonly occurs in irritable bowel syndrome in patients experiencing nervousness or under stress conditions. However, no reports on the effects of AS or ASRE on GI motility have been identified. Therefore, as our primary study aim, we determined whether ASRE fundamentally alters contractile smooth muscle function using an isolated mouse ileum model and an organ bath system. Furthermore, based on the in vitro experimental results, we assessed the effects of ASRE on GI motility using the GI transit method in normal mice and a carbachol (CCh)-induced diarrhea mouse model.⁴⁾ This study may provide new strategies to treat abnormal GI mobility especially diarrhea symptoms, resulting in an increase in the parasympathetic nerve activities.

MATERIALS AND METHODS

Animals

Male ICR mice (weighing 25–40 g) were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and housed for at least 1 week in a temperature controlled environment (22–24°C) at 50 ± 10% humidity under a 12-h light/dark cycle before starting the experiments. They were provided standard chow (CE-2; Clea Japan Inc., Tokyo, Japan) and water ad libitum. All protocols involving animals were approved by the animal ethics committee (P-12-2018-04-A) and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Mukogawa Women’s University, Japan.

Materials

ASRE dry powder was provided by Sun Chlorella Co. (Kyoto, Japan, Lot No. 8142, 100% AS), which is the same product we previously used.²⁴⁾ The dried root tips (fresh weight: 10 kg) of AS from Heilongjiang, China were extracted once with water for 3 h at 80°C. The aqueous layer was evaporated in vacuo, yielding approximately 250 g of powder using a spray-drying method. The herbal specimen was authenticated by Emeritus Professor Sansei Nishibe of the Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Health Science University of Hokkaido.²³⁾ The ASRE composition in 100 g was 101.4 mg isofraxidine, 625.2 mg eleutheroside E (acanthoside D), 325.2 mg eleutheroside B, 95.2 mg eleutheroside B1, and 829.5 mg chlorogenic acid measured using HPLC and an octadecylsilyl column, according to the manufacturer's instruction. ASRE was dissolved in distilled water containing 0.5% dimethyl sulfoxide for in vitro studies and only in distilled water for in vivo studies.

Acetylcholine (ACh) was purchased from Daiichi Sankyo Co., Ltd. (Tokyo, Japan). Atropine, CCh, glibenclamide (Gli), N^G-nitro-L-arginine methyl ester (L-NAME), and propranolol were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Charcoal, and Arabic gum were obtained from Wako Pure Chemical Corporation (Osaka, Japan). Tetraethylammonium chloride (TEA), and potassium chloride (KCl) were acquired from Nacalai Tesque (Kyoto, Japan). Gli was dissolved in 100% dimethyl sulfoxide at a final concentration of 10⁻² M. TEA was dissolved in Krebs–Henseleit (Krebs) solution and all other chemicals were dissolved in distilled water.

Krebs solution (118.4 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂·2H₂O, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄·7H₂O, 25.0 mM NaHCO₃, 11.1 mM glucose bubbled with 95% O₂/5% CO₂ gas) was prepared and used for the organ bath experiments.

Effects of ASRE on Isolated Mouse Ileum

Contractile and relaxing ileal responses were measured using an organ bath as previously described.²⁵⁾ Mice fasted for 24 h, and each approximately 10-mm long ileal segment was removed from the upper position (30 mm or more) of the ileum-cecum junction, and immediately suspended in a 10-mL organ bath chamber filled with Krebs solution, as described above, with a resting tension of 0.5 g. Before starting the experiments, ACh (10⁻⁶ M) was added to the bath and this procedure was performed twice. Between all measurements, the fresh Krebs solution was changed three times every 10 min and stabilized for 30 min.

In the experiments, ASRE (10⁻⁵–10⁻² g/mL) or ACh (10⁻¹⁰–3 × 10⁻⁵ M) was cumulatively added to the organ bath, and the first reaction (control) was measured. After the stabilization period of 30 min, the second reaction was performed using ASRE (10⁻⁵–10⁻² g/mL) in the presence or absence of inhibitors: atropine (3 × 10⁻⁶ M), L-NAME (10⁻⁴ M, 20 min), propranolol (10⁻⁵ M, 10 min), Gli (10⁻⁵ M, 10 min), or TEA (1 mM, 30 min). By using ACh (10⁻¹⁰–3 × 10⁻⁵ M) or KCl (40 mM), we examined the effect of ASRE on the contractile response induced by ACh or KCl. After a stabilization period of 30 min, the second reaction with ACh (10⁻¹⁰–3 × 10⁻⁵ M) or KCl (40 mM) in the presence or absence of ASRE (3 × 10⁻⁴ g/mL, 3 min) or atropine (3 × 10⁻⁶ M, 3 min), respectively was measured. The treatment condition of each inhibitor was decided according to preliminary experiments and previous reports.^25–28)

The contraction or relaxation in the presence of each inhibitor was calculated in relation to the maximum contractile force in the first reaction, which corresponded to 100%. The contractile response to drug concentration curves were analyzed using nonlinear curve fitting, and the negative log half maximal effective concentration (EC₅₀) and maximum response (E_max) were calculated using GraphPad Prism^® software (ver. 5.0, GraphPad, Inc., La Jolla, CA, U.S.A.). Contraction and relaxation data were analyzed by dividing the force generated by ASRE (g tension) by the weight of the wet tissue (g).

Effects of ASRE on Mouse GI Transit

To investigate effects of oral ASRE administration on GI transit in mice, we used the GI transit method following previously published guidelines.^4,25) The mice were starved for 24 h before the experiment but water was available ad libitum. Saline (vehicle) or CCh (1 mg·kg⁻¹) solution was subcutaneously injected. After 15 min, ASRE (900 mg·kg⁻¹) or distilled water (vehicle) was administered via oral gavage. Atropine (1 mg/kg) was subcutaneously injected in CCh-induced model mice. After 30 min, a charcoal meal (5% charcoal/10% Arabic gum) was orally administered at 10 mL·kg⁻¹ body weight. Finally, after 20 min, the mice were sacrificed by cervical dislocation under anesthesia with sodium pentobarbital (65 mg·kg⁻¹, intraperitoneally (i.p.)), and the small intestines were carefully isolated and immediately rinsed with ice-cold Krebs solution. GI transit of the charcoal meal was calculated for each mouse as the percentage of the distance traveled by the charcoal meal relative to the whole length of the small intestine.

Additionally, a 4-d treatment with oral ASRE administration was performed. ASRE (900 mg·kg⁻¹·d⁻¹) or distilled water was orally administered once a day for 3 d. After starving the mice for 24 h, they received the treatment followed by evaluation of the GI transit on day 4.

Based on the guide for dose conversion between animals and humans using body surface area,²⁹⁾ the ASRE dose of 900 mg·kg⁻¹·d⁻¹ was estimated to be approximately equal to the 6 g·d⁻¹ (the conventional human dose) of ASRE in humans provided by the manufacturer.

Data Analysis

All data are expressed as the means ± standard error of the mean (S.E.M.). Statistical analyses of contraction or relaxation data of the isolated mouse ilea were performed using the Student’s t-test or one-way ANOVA, followed by Dunnett’s test for multiple comparisons. GI transit data were analyzed using a one-way ANOVA followed by Bonferroni’s multiple comparison test. Values were considered significant when p < 0.05. Statistical analyses were performed using GraphPad Prism^® software.

RESULTS

Effects of ASRE on Isolated Mouse Ileal Motility

ASRE (10⁻⁵–10⁻² g/mL, Fig. 1A) and ACh (10⁻¹⁰–3 × 10⁻⁵ M, Fig. 1B) induced contractions in a dose-dependent manner. The maximum contractile force induced by ASRE was approximately half of that induced by ACh (Table 1). In the presence of atropine (3 × 10⁻⁶ M, 3 min), a non-selective muscarinic receptor antagonist, ASRE-induced contractions at 10⁻⁵–10⁻³ g/mL disappeared, while relaxation responses were observed at 3 × 10⁻³ and 10⁻² g/mL (Fig. 2). L-NAME did not affect the atropine-induced relaxation in the presence of ASRE (data not shown). Furthermore, neither the ATP-sensitive K⁺ channel blocker, Gli (10⁻⁵ M, 10 min), nor the BK_ca channel blocker, TEA (1 mM, 30 min), affected ASRE-induced relaxations (data not shown).

Fig. 1. Contractile Responses Induced by Acanthopanax senticosus Root Extract (ASRE, A) and Acetylcholine (ACh, B) in Isolated Mouse Ileum

Increasing concentrations of ASRE or ACh were cumulatively added to organ bath. Each point represents mean ± S.E.M., n = 4.

Table 1. Parameters of Contractile Responses to Acanthopanax senticosus Root Extract (ASRE) or Acetylcholine (ACh) in Isolated Mouse Ileum

	−log EC50 (g/mL or M)	Maximum contractile force (g tension/g weight of wet tissue)
ASRE	3.32 ± 0.14	7.76 ± 0.95**
ACh	6.82 ± 0.06	15.6 ± 1.5

The negative log EC₅₀ value was calculated from each concentration–response curve. The maximum contractile force was calculated according to the contractile strength (g tension) and the sample weight (g). Values are means ± S.E.M., n = 4. ** p < 0.01 (Student’s t-test)

Fig. 2. Effects of Atropine on Responses Induced by Acanthopanax senticosus Root Extract (ASRE) in Isolated Mouse Ileum

ASRE was cumulatively added in the presence (hatched column) or absence (solid column) of atropine (3 × 10⁻⁶ M, 3 min). Each point represents mean ± S.E.M., n = 5.

ACh-induced contractions of the isolated mouse ileum decreased in the presence of 3 × 10⁻⁴ g/mL ASRE (Fig. 3A and Table 2). In contrast, the β-blocker, propranolol, had no effect on the inhibition of the ACh-induced contractions by ASRE (Fig. 3B). Furthermore, 3 × 10⁻⁴ g/mL ASRE also reduced contractions induced by 40 mM KCl, while atropine (3 × 10⁻⁶ M) had no effect (Fig. 3C).

Fig. 3. Effects of Acanthopanax senticosus Root Extract (ASRE) on Contractions Induced by Acetylcholine (ACh, A and B) and Potassium Chloride (KCl, C) in Isolated Mouse Ileum

ACh was cumulatively added to the organ bath in the presence (solid diamond, A and B) or absence (open diamond) of ASRE (3 × 10⁻⁴ g/mL, 3 min) (A), with (open hexagon) or without (solid diamond) propranolol (10⁻⁵ M, 10 min) (B). KCl (40 mM) was added to the organ bath in the presence of ASRE (3 × 10⁻⁴ g/mL, 3 min, solid column), atropine (3 × 10⁻⁶ M, 3 min, hatched column), or vehicle (0.0005% dimethyl sulfoxide, open column) (C). Values represents the means ± S.E.M., n = 4. ** p < 0.01 (Student’s t-test or one-way ANOVA followed by Dunnett’s test for multiple comparisons).

Table 2. Effects of Acanthopanax senticosus Root Extract (ASRE) on Acetylcholine (ACh)-Induced Contractile Response in Isolated Mouse Ileum

	−log EC50 (M)	Emax (% of control)
Absence of ASRE	7.03 ± 0.10	124.5 ± 6.7
Presence of ASRE	6.92 ± 0.10	97.5 ± 6.2*

The negative log EC₅₀ and E_max values were calculated from each concentration–response curve. ASRE (3 × 10⁻⁴ g/mL, 3 min) was added to the organ bath, followed by ACh. Values are means ± S.E.M., n = 4. * p < 0.05 (Student’s t-test).

Effects of Single and 4-d Oral Administration of ASRE on GI Transit of Charcoal Meal in Mice

We examined the effects of a single oral administration of 900 mg·kg⁻¹ ASRE on GI transit in normal mice and a CCh-induced diarrhea mouse model (Fig. 4A). Oral administration of ASRE for 30 min did not affect the distance traveled by the charcoal meal in normal mice. On the contrary, induction of diarrhea in mice with 1 mg·kg⁻¹ CCh, increased GI transit of the charcoal meal and this effect was decreased by ASRE administration. A beneficial effect was observed following treatment with 1 mg·kg⁻¹ atropine, similar to that observed in previous studies.⁴⁾

Fig. 4. Effects of 1- (A) and 4- (B) Day Oral Administration of Acanthopanax senticosus Root Extract (ASRE, 900 mg/kg) on Charcoal Gastrointestinal (GI) Transit in Mice

After saline (3 mL/kg, s.c.) or carbachol (CCh, 1 mg/kg, s.c.) was injected for 15 min, distilled water (H₂O, p.o.), ASRE (p.o.), or atropine (1 mg/kg, s.c.) was provided for 30 min. Finally, 5% charcoal/10% Arabic gum was orally applied for 20 min and GI transit was evaluated in mice. Values are the means ± S.E.M., n = 5 (A) and 3–4 (B). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA followed by Bonferroni’s multiple comparison test).

Furthermore, we determined whether the effect of ASRE on normal mice or the CCh-induced diarrhea mouse model changed when the treatment was extended from 1 to 4 d (Fig. 4B). Similar to the effects of single-dose ASRE administration (Fig. 4A), multiple administrations of ASRE did not affect charcoal transport in the normal mice, but inhibited the increased GI transit in the CCh-induced diarrhea model mice (Fig. 4B).

DISCUSSION

This study demonstrated that ASRE induced dose-dependent contractions of the isolated mouse ileum and that ACh-like muscarinic receptor agonism may be involved in the contractions. In contrast, in the presence of a muscarinic antagonist, atropine, ASRE directly relaxed the mouse ileum. Furthermore, oral administration of 900 mg·kg⁻¹ ASRE, which is almost the daily dose in humans, did not affect GI transit of the charcoal meal in normal mice. However, ASRE reduced the increased GI transit observed with CCh-induced diarrhea. This favorable action was observed only in the diarrhea mouse model after 1 or 4 d of treatment. These results suggested that ASRE may exhibit antidiarrheal activity by modulating GI motility.

ASRE exerted a dual action in the mouse ileum in the in vitro experiments and induced contractions to a maximum degree that was almost half of that caused by ACh. We initially measured the amount of ACh contained in ASRE using the ACh assay, as previously described,²⁵⁾ and we determined that the concentration in ASRE was below the detection limit. Since the ASRE-induced contraction was completely antagonized by atropine, the ASRE probably contained an ACh-like substance, which seemed to be the main constituent that caused the ileal contraction although its concentration was very low. In contrast, unexpectedly, ASRE relaxed the mouse ilea in the presence of atropine, which antagonized muscarinic receptor(s). To further experimentally elucidated the underlying mechanism of ASRE and found that it significantly reduced ACh- and high K⁺-induced contractions, although atropine was not effective in the second scenario. These findings indicate that the ASRE may have directly relaxed the ileal smooth muscle, and did not contain an atropine-like substance. Our previous findings demonstrated that ASRE induced endothelial nitric oxide (NO)-independent vasorelaxation of the rat thoracic aorta²⁴⁾ and raised the possibility that K⁺ channel opener(s) or Ca²⁺ channel inhibitor(s) in ASRE mediated the mechanisms underlying relaxation of the vascular smooth muscle. In fact, in the intestine, it has been revealed that adenosine plays as an inhibitory modulatory role in the contractility of the mouse ileal longitudinal muscles by opening large (BK_Ca) and small (SK_Ca) conductance Ca²⁺-activated K⁺ channels.²⁸⁾ However, the present study demonstrated that both ATP-sensitive K⁺ channel blocker (Gli) and non-specific K⁺ channel blocker (TEA) did not affect the ASRE-induced relaxations under atropine treatment in the mouse ileum. Additionally, propranolol, a non-selective β receptor antagonist, also did not affect the ASRE-induced relaxation. These findings could preclude the possible involvement of NO produced by ASRE and the presence of β agonist(s) or K⁺ channel opener(s) in the relaxation of the mouse ileum by ASRE. Another possible mechanism underlying the effect of ASRE is blockade of Ca²⁺ channels in the ileal smooth muscle, leading to inhibition of the contractile response.

Since ASRE showed dual opposing contraction and relaxation functions in the isolated mouse ileum, it was difficult to predict whether ASRE improved constipation or diarrhea. We previously demonstrated that although royal jelly contained a high concentration of ACh and induced contractions of the isolated mouse ileum in vitro, oral administration did not significantly alter GI motility in both normal mice and a loperamide-induced constipation model mouse.²⁵⁾ From these findings, we concluded that ACh-like substances in ASRE did not increase in vivo ileal mouse motility. In contrast, we suspected that ASRE-induced relaxation could affect GI functions. Therefore, we studied the effects of oral administration of ASRE on normal mice and a CCh-induced diarrhea mouse model using the GI transit method. Oral administration of ASRE to normal mice at a dose of 900 mg·kg⁻¹·d⁻¹, which is equivalent to the daily 6 g human dose, had no effect on GI transit of the charcoal meal after a 1- or 4-d treatment. In contrast, a single administration of ASRE to the CCh-induced diarrhea mouse model decreased the enhanced intestinal motility. Furthermore, 4-d administration of ASRE improved intestinal motility, which was enhanced by CCh and its efficacy/potency did not differ from that observed after the 1-d treatment. Importantly, the ASRE-induced decrease in GI motility was specific to conditions where the intestinal motility was enhanced, as a 1- or 4-d ASRE administration did not alter the intestinal motility in mice under normal condition. In contrast, a possible transient decrease in blood pressure on the day 1 of the treatment was observed, as previously reported in rats.²⁴⁾ Furthermore, possible drug interactions should be considered when ASRE is combined with other drugs and supplements. A previous study reported that AS extract suppressed the activities of P-glycoprotein and peptide transporters in a non-competitive manner in caco-2 cells,^30,31) whereas it did not affect the activity of CPY 2D6 or CYP3A4 when administered at the usual recommended dose to normal volunteers.³⁰⁾

In the present study, we did not identify the active component of ASRE, especially the component that caused ileum relaxation. However, chlorogenic acid is one of the major components of the ASRE, according to the manufacturer’s report. Chlorogenic acid has been shown to elicit an antispasmodic effect on the rat jejunum and guinea pig ileum^32,33) and dose-dependently inhibit CCh-induced contractions of the mouse bladder.³⁴⁾ In contrast, chlorogenic acid was reported to slightly increase contractile activity of the guinea pig ileum.³⁵⁾ However, there are no other reports about other components that contract or relax the ileum. Therefore, further investigations of the active components of ASRE mediating the relaxant effects on the ileum and their underlying mechanisms are necessary.

CONCLUSION

In conclusion, the ASRE investigated in this study caused in vitro contraction by interacting with muscarinic ACh receptors and direct relaxation of mouse ileal smooth muscle. In addition, 1- or 4-d oral administration of ASRE had no effect on GI transit in mice under normal conditions, whereas it decreased the enhanced intestinal motility under CCh-induced diarrhea conditions. These findings indicated that ASRE did not cause serious symptoms such as constipation under normal conditions, but it improved enhanced intestinal motility with symptoms such as diarrhea. Further investigations of the active ingredients in ASRE that contribute to ileal relaxation and the mechanisms underlying this effect are needed. However, the results of this study suggest that ASRE has potential as a new strategy for the treatment of abnormal GI motility especially diarrhea symptoms, resulting in an increase in the parasympathetic nerve activities.

Acknowledgments

The authors express their sincere gratitude to Ms. Kana Morikawa, Ms. Miho Shimari, Ms. Moeka Norii, and Ms. Maho Mizuno for technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

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