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Keywords:

  • Boswellia serrata;
  • boswellic acids;
  • intestinal motility;
  • spasmolytic activity;
  • diarrhoea;
  • calcium channels

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Clinical studies suggest that the Ayurvedic plant Boswellia serrata may be effective in reducing diarrhoea in patients with inflammatory bowel disease. In the present study, we evaluated the effect of a Boswellia serrata gum resin extract (BSE) on intestinal motility and diarrhoea in rodents.
  • 2
    BSE depressed electrically-, acetylcholine-, and barium chloride-induced contractions in the isolated guinea-pig ileum, being more potent in inhibiting the contractions induced by acetylcholine and barium chloride.
  • 3
    The inhibitory effect of BSE on acetylcholine-induced contractions was reduced by the L-type Ca2+ channel blockers verapamil and nifedipine, but not by the sarcoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid, by the phosphodiesterase type IV inhibitor rolipram or by the lipoxygenase inhibitor zileuton.
  • 4
    3-acetyl-11-keto-β-boswellic acid, one of the main active ingredients of B. serrata, inhibited acetylcholine-induced contractions.
  • 5
    BSE inhibited upper gastrointestinal transit in croton oil-treated mice as well as castor oil-induced diarrhoea. However, BSE did not affect intestinal motility in control mice, both in the small and in the large intestine.
  • 6
    It is concluded that BSE directly inhibits intestinal motility with a mechanism involving L-type Ca2+ channels. BSE prevents diarrhoea and normalizes intestinal motility in pathophysiological states without slowing the rate of transit in control animals. These results could explain, at least in part, the clinical efficacy of this Ayurvedic remedy in reducing diarrhoea in patients with inflammatory bowel disease.

British Journal of Pharmacology (2006) 148, 553–560. doi:10.1038/sj.bjp.0706740


Abbreviations:
BSE

Boswellia serrata gum resin extract

DMSO

dimethyl sulphoxide

PDE

phosphodiesterase

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Boswellia serrata Roxb (Fam Burseraceae), also called Indian olibanum, is a moderate to large branching tree native of India, North Africa and the Middle East. A gum resin, obtained tapping the tree trunk, is widely used in Ayurvedic medicine for the treatment of inflammatory diseases, including those affecting the gastrointestinal tract (e.g. diarrhoea, dysentery, and inflammatory bowel disease). The anti-inflammatory activity of B. serrata gum resin has been confirmed by experimental and clinical studies (Capasso et al., 2003). For example, extracts of B. serrata gum resin: (i) display a marked anti-inflammatory activity in carrageenan-, dextran-, and papaya-latex-induced models of inflammation in rodents (Singh & Atal, 1986; Gupta et al., 1992), (ii) reduce the infiltration of polymorphonuclear leucocytes in carrageenan-induced pleurisy (Sharma et al., 1988), and (iii) inhibit leukotriene synthesis from arachidonic acid in rat peritoneal polymorhonuclear leukocytes (Ammon et al., 1991). Boswellic acids (β-boswellic acid, 3-acetyl-β-boswellic acid, 11-keto-β-boswellic acid and 3-acetyl-11-keto-β-boswellic acid) have been suggested to be the active constituents of this herbal drug (Safayhi et al., 1992). These compounds are specific, nonredox inhibitors of 5-lipoxygenase without affecting 12-lipoxygenase and cyclooxygenase activities (Ammon et al., 1993; Wildfeuer et al., 1998; Safayhi et al., 2000).

A purified extract of the resin is actually used in modern herbal preparations to treat a number of inflammatory conditions including inflammatory bowel disease. Clinical studies have shown that B. serrata gum resin is effective in patients with ulcerative colitis (grades II and III). Treatment with this herbal drug is associated to improvement of a number of parameters of the pathology, including stool consistency and frequency (Gupta et al., 1997; 2001; Gerhardt et al., 2001) and it has been considered superior over mesalazine in terms of a benefit-risk evaluation (Gupta et al., 1997).

As a result of the clinically established symptomatic improvement of inflammatory bowel disease symptoms (including the reduction of the diarrhoea) seen under treatment with B. serrata and its traditional use in Ayurvedic medicine as antidiarrhoeal agent, we investigated the effect and the mode of action of this herbal drug on intestinal motility, both in vitro and in vivo. We also evaluated the effect of 3-acetyl-11-keto-β-boswellic acid, one of the main active ingredients of B. serrata.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals

Male ICR mice (20–22 g) and New Zealand guinea-pigs (300–450 g) were supplied by Harlan Nossan Italy, Corezzana, MI, U.S.A. All animals, used after 1 week of acclimation (temperature 23±2°C; humidity 60%), had free access to water and food. All experiments on animals complied with the Italian D.L. no. 116 of 27 January 1992 and associated guidelines in the European communities Council Directive of 24 November 1986 (86/609/ECC).

In vitro experiments

Guinea-pigs were killed by asphyxiation with CO2 and segments (2–3 cm) of the terminal ileum were removed, flushed of luminal contents, and placed in Krebs' solution (composition in mM: NaCl 119, KCl 4.75, KH2PO4 1.2, NaHCO3 25, MgSO4 1.5, CaCl2 2.5, and glucose 11). The isolated organ was set up (in such a way to record contractions from the longitudinal axis) in an organ bath filled warm (37°C) aerated (95% O2 : 5% CO2) Krebs' solution. The tissues were connected to an isotonic transducer (load 0.5 g) connected to a PowerLab data-acquisition system (Ugo Basile, Comerio, Italy). At the beginning of each experiment, the ileum was stimulated with acetylcholine (10−3 M) in order to obtain a maximal contraction (100% contraction). After a minimal 1-h equilibration period, the tissues were subjected to electrical field stimulation (EFS, 10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration using a multiplexing pulse booster by Ugo Basile, Milan, Italy) via a pair of platinum electrodes (situated at a distance of 1.5 cm) placed around the intestine or stimulated with spasmogens, namely acetylcholine (10−6 M) or barium chloride (10−4 M). The concentrations of acetylcholine and barium chloride gave a contractile response which was similar in amplitude to that of EFS. Acetylcholine and barium chloride were added to the bath and left in contact with the tissue for 30 s and then washed out. The interval between each stimulation was 20 min. After at least three stable control contractions, the contractile responses were repeated in the presence of increasing (noncumulative) concentrations of B. serrata gum resin extract (BSE, 0.001–3 mg ml−1) added 20 min before the contacting stimulus (i.e. after washing the tissue). Preliminary experiments showed that a 20 min contract time was sufficient for BSE to achieve the maximal inhibitory effect.

In some experiments, the effect of BSE on acetylcholine-induced contractions was evaluated in the presence of verapamil (10−6 M), nifedipine, (10−6 M), cyclopiazonic acid (10−5 M), rolipram (10−6 M), or zileuton (10−5 M) (contact time: 20 min for each drug). The concentrations of verapamil, nifedipine, cyclopiazonic acid, rolipram, and zileuton were selected on the basis of previous published work (Uyama et al., 1992; Izzo et al., 1999; Aronsson & Holmgren, 2000; Lis-Balchin & Hart, 2002). The presence of such inhibitors/antagonists did not affect the reproducibility and the stability of the contractions induced by acetylcholine. In another set of experiments, we evaluated the effect of 3-acetyl-11-keto-β-boswellic acid (2 × 10−7–2 × 10−4 M, contact time: 20 min per concentration) on acetylcholine (10−6 M)-induced contractions.

Finally, some experiments were performed using the mouse terminal ileum (1–1.5 cm). This tissue was stimulated by acetylcholine (10−6 M) and the effect of BSE (1–1000 μg ml−1) was evaluated. The experimental conditions and protocol of drugs administration were the same as described for the guinea-pig ileum.

In vivo experiments

Chronic intestinal inflammation

Inflammation was induced as previously described (Pol & Puig, 1997; Izzo et al., 2000). Briefly, two doses of croton oil (20 μl mouse−1) in two consecutive days were orally administered to mice and four days after the first administration of croton oil, upper gastrointestinal transit of mice was measured. This time was selected on the basis of a previous work (Pol & Puig, 1997), which reported that maximal inflammatory response occurred 4 days after the first treatment.

Upper gastrointestinal transit

Upper gastrointestinal transit was measured in control and in croton oil-treated mice. A black marker (0.1 ml 10 g mouse−1; 10% charcoal suspension in 5% gum Arabic) was administered orally to assess gastrointestinal transit as previously described (Pol & Puig, 1997; Izzo et al., 2001). After 20 min the mice were killed by asphyxiation with CO2 and the gastrointestinal tract removed. The distance travelled by the marker was measured and expressed as a percentage of the total length of the small intestine from pylorus to caecum. BSE (100–400 mg kg−1) or vehicle (carboxymethylcellulose 1%) was given orally 60 min before charcoal administration, both to control mice and to mice with chronic inflammation. In some experiments, we evaluated the effect of atropine (1 mg kg−1), used as a reference drug, on motility in control mice.

Colonic propulsion

Distal colonic propulsion was measured as previously described (Broccardo et al., 1998; Pinto et al., 2002). A single 3-mm glass bead was inserted 2 cm into the distal colon of each mouse with the aid of a catheter and the time to expulsion of the glass bead was determined for each animal. BSE (100–400 mg kg−1), atropine (1 mg kg−1, used as a reference drug), or vehicle (carboxymethylcellulose 1%) were given orally 60 min before glass bead insertion.

Castor oil-induced diarrhoea

Diarrhoea was induced by oral administration of castor oil (0.2 ml mouse−1) to mice fasted for a night. BSE (100–400 mg kg−1) or vehicle (carboxymethylcellulose 1%) were given orally 60 min before cathartic administration. At 2 h after dosing with castor oil the individual mouse cages were inspected (by an observer unaware of the particular treatment) for the presence of unformed water fecal pellets; their absence was recorded as a positive result, indicating protection from diarrhoea at that time (Capasso et al., 1994). In some experiments, the effect of BSE (100–400 mg kg−1) on castor oil-induced diarrhoea was evaluated in mice pretreated (i.p.) with zileuton (35 mg kg−1, 30 min before BSE).

Drugs

Drugs used were: castor oil, acetylcholine chloride, barium chloride, verapamil, nifedipine, cyclopiazonic acid, rolipram, atropine, tetrodotoxin, polyethylene glycol, carboxymethylcellulose (all from Sigma, Milan, Italy), zileuton (Sequoia Research Products Ltd, U.K.), and 3-acetyl-11-keto-β-boswellic acid (LGC Promochem GmbH, Germany); B. serrata gum resin hydroalcoholic extract (BSE, standardized to contain 95% boswellic acids) was a gift from Carlo Sessa, Milan, Italy. Acetylcholine, barium chloride, atropine, and tetrodotoxin were dissolved in distilled water; zileuton, verapamil, nifedipine, cyclopiazonic acid, and 3-acetyl-11-keto-β-boswellic acid in dimethyl sulphoxide (DMSO). Rolipram was dissolved in DMSO to give 10−5 M stock solution and subsequent dilutions were made in distilled water. BSE was suspended in carboxymethylcellulose 1% (for in vivo experiments) or in polyethylene glycol (for in vitro experiments). Drugs were added in volumes <0.01% in vitro and given in the amount of 0.01 ml mouse−1 (DMSO) or 0.1 ml mouse−1 (carboxymethylcellulose) in vivo. The drug vehicles had no effect on the responses under study, both in vitro and in vivo.

Statistics

Data are mean±s.e.m. Comparisons between two sets of data were made by Student's t-test for paired data. When multiple comparisons against a single control were made, one-way analysis of variance was used, followed by Turkey–Kramer multiple comparisons test. Analysis of variance (two way) was used to compare different cumulative concentration–effect curves.

Diarrhoea was expressed as total score and the χ2 test was used to determine the significance between groups. A P-value <0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In vitro experiments

The contractile responses of guinea-pig ileum to EFS reached 55.3±4.56% (n=6) of the contraction produced by acetylcholine 10−3 M. This concentration of acetylcholine produced a maximal contractile response in the ileum (100% contraction). EFS-induced contractions were abolished by tetrodotoxin (3 × 10−7 M) or atropine (10−6 M), thus suggesting that these contractions were mediated by the release of acetylcholine from enteric nerves. However, tetrodotoxin did not modify the contractions induced by either acetylcholine (10−6 M) or barium chloride (10−4 M) (data not shown).

BSE (1–1000 μg ml−1) significantly and in a concentration-dependent manner, inhibited the contractions induced by acetylcholine, barium chloride or by EFS (Figure 1). BSE was significantly (P<0.001) more active in inhibiting the contractions induced by acetylcholine (or barium chloride) than the contractions induced by EFS (Figure 1). Figure 2 reports a typical trace showing the inhibitory effect of BSE on acetylcholine-induced contractions.

image

Figure 1. Inhibitory effect of B. serrata gum resin extract (BSE, 1–1000 μg ml−1) on the contractile response induced by exogenous acetylcholine (ACh, 10−6 M), barium chloride (BaCl2, 10−4 M), or electrical field stimulation (EFS, 10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration) in the isolated guinea-pig ileum. Each point represents mean±s.e.m. of six to eight experiments. ***P<0.001 vs ACh (significance between the two dose–response curves).

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image

Figure 2. Typical trace showing inhibitory effect of B. serrata gum resin extract (BSE, 1–1000 μg ml−1) on contractions produced by acetylcholine in isolated guinea-pig ileum. Dots indicate contractions induced by acetylcholine (10−6 M), arrows indicate the administrations of BSE in the organ bath.

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Verapamil (10−6 M) and nifedipine (10−6 M), but not cyclopiazonic acid (10−5 M), significantly reduced the inhibitory effect of BSE on acetylcholine-induced contractions (Figure 3). Rolipram (10−6 M) and zileuton (10−5 M) did not modify the effect of the extract on acetylcholine-induced contractions (rolipram % of inhibition: BSE 1 μg ml−1 3.93±2.53, BSE 3 μg ml−1 5.13±3.16, BSE 10 μg ml−1 15.9±4.23, BSE 30 μg ml−1 29.2±5.18, BSE 100 μg ml−1 52.3±6.34, BSE 300 μg ml−1 76.6±3.51, BSE 1000 μg ml−1 96.0±3.02; zileuton % of inhibition: BSE 1 μg ml−1 3.01±1.89, BSE 3 μg ml−1 4.68±2.58, BSE 10 μg ml−1 18.9±4.05, BSE 30 μg ml−1 30.14±5.01, BSE 100 μg ml−1 55.9±6.15, BSE 300 μg ml−1 80.1±3.92, BSE 1000 μg ml−1 97.1±3.11). When given alone (i.e. in absence of BSE) verapamil (10−6 M), nifedipine (10−6 M) and cyclopiazonic acid (10−5 M) reduced (verapamil: 45.51±5.41% reduction, P<0.001; nifedipine: 43.26±3.45% reduction, P<0.001; cyclopiazonic acid: 54.13±6.22, P<0.001, n=8 for each drug) the contractions induced by acetylcholine. By contrast, rolipram (10−6 M) increased (47.83±8.98% increase, P<0.01) while zileuton (10−5 M) did not modify acetylcholine-induced contractions. Verapamil and nifedipine, at a concentration higher than 10−6 M (i.e. 3 × 10−6 M) did not produce a further inhibitory effect on both acetylcholine-induced contractions and BSE antispasmodic effect (data not shown). 3-acetyl-11-keto-β-boswellic acid (2 × 10−7–2 × 10−4 M), significantly and in a concentration dependent manner, inhibited the contractile response elicited by acetylcholine (Figure 4). BSE (1–1000 μg ml−1) and 3-acetyl-11-keto-β-boswellic acid (2 × 10−7–2 × 10−4 M) had no effect on the baseline mechanical activity of the intestine (data not shown).

image

Figure 3. Acetylcholine-induced contractions in isolated guinea-pig ileum: effect of B. serrata gum resin extract (BSE, 1–1000 μg ml−1) alone (vehicle) or in the presence of verapamil (10−6 M), nifedipine (10−6 M) or cyclopiazonic acid (10−5 M). Each point represents mean±s.e.m. of six to eight experiments. **P<0.01 and ***P<0.001 vs vehicle (significance between the two dose–response curves).

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image

Figure 4. Inhibitory effect of 3-acetyl-11-keto-β-boswellic acid (2 × 10−7–2 × 10−4 M) on the contractions induced by acetylcholine (10−6 M) in the isolated guinea-pig ileum. Each point represents mean±s.e.m. of six to eight experiments.

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Finally, BSE also inhibited the contractions induced by acetylcholine in the mouse ileum (percentage of inhibition: BSE 1 μg ml−1 3.54±1.17, BSE 3 μg ml−1 7.02±1.96, BSE 10 μg ml−1 20.9±4.02, BSE 30 μg ml−1 32.8±4.12, BSE 100 μg ml−1 51.3±5.10, BSE 300 μg ml−1 79.8±4.23, BSE 1000 μg ml−1 98.1±3.44; P<0.05 for the 30 and 100 μg ml−1 concentrations, P<0.01 for the 300 μg ml−1 concentration and P<0.001 for the 1000 μg ml−1 concentration, n=6).

In vivo experiments

Upper gastrointestinal transit and colonic propulsion in control mice

Oral administration of BSE (100–400 mg kg−1) had no effect on motility, both in the upper gastrointestinal tract (Figure 5a) and in the large intestine (Figure 5b). By contrast, atropine (1 mg kg−1, used as a reference drug) inhibited motility both in the upper gastrointestinal tract and in the large intestine (Figure 5a and b).

image

Figure 5. Effect of B. serrata gum resin extract (BSE, 100–400 mg kg−1) and atropine (AT, 1 mg kg−1) on upper gastrointestinal transit (a) and colonic propulsion in mice (b). Results are mean±s.e.m. of 10–12 animals for each experimental group. **P<0.01 vs control.

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Upper gastrointestinal transit in croton oil-treated mice

According to previous studies (Pol & Puig, 1997; Capasso et al., 2001), administration of croton oil produced a significant increase (P<0.01) in upper gastrointestinal transit (Figure 6). BSE (100–400 mg kg−1) counteracted the increase in motility induced by croton oil (Figure 6). The effect was significant starting from the 200 mg kg−1 oral dose.

image

Figure 6. Effect of B. serrata gum resin extract (BSE, 100–400 mg kg−1) on upper gastrointestinal transit in mice treated with croton oil (20 μl mouse−1). Results are mean±s.e.m. of 10–12 animals for each experimental group. #P<0.01 vs control; *P<0.05 vs croton oil.

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Castor oil-induced diarrhoea

At 2 h after castor oil administration (0.2 ml mouse−1) mice produced copious diarrhoea. BSE significantly (P<0.01) reduced castor oil-induced diarrhoea in a dose-related manner (Figure 7). Consistent with the experiments in mice treated with croton oil, a significant inhibitory effect was achieved for the 200–400 mg kg−1 oral doses. Zileuton (35 mg kg−1) did not modify castor oil-induced diarrhoea (% of mice with diarrhoea: castor oil 91.7; castor oil+zileuton 93.3; n=12–15 for each experimental group, P>0.2) or the antidiarrhoeal effect of BSE (% of mice with diarrhoea: castor oil 91.7; castor oil+BSE 200 mg kg−1 63.63; castor oil+zileuton+BSE 60.0; n=12–15 for each experimental group, P>0.2).

image

Figure 7. Effect of B. serrata gum resin extract (BSE, 100–400 mg kg−1) on castor oil-induced diarrhoea. The effect of BSE was assessed 2 h after castor oil (0.2 ml mouse−1) administration (n=10–12). *P<0.05 and **P<0.01 vs control.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

B. serrata is an Ayurvedic remedy widely employed for the treatment of diarrhoea and inflammatory bowel disease. Suppression of leukotriene synthesis via inhibition of 5-lipoxygenase has been considered to be the main mechanism underlying its beneficial effect in intestinal inflammatory disease (Ammon, 1996). The present study provides evidence that BSE inhibits intestinal motility which may contribute to the clinical efficacy of this herbal drug in normalizing motility changes associated to inflammatory bowel disease.

We have shown that BSE produced a concentration-dependent inhibition of both acetylcholine- and electrical field stimulation-evoked contractions in the isolated guinea-pig ileum. Moreover, BSE preferentially inhibited the contractions induced by acetylcholine (which are due to a direct activation of muscarinic receptors located on smooth muscles) rather than the contractions elicited by EFS (which are mediated by the release of acetylcholine from myenteric nerves). These results indicate that BSE (i) could release from neural or non-neural sources endogenous substances which have an excitatory effect at prejunctional level and, more importantly, that (ii) exerts an antispasmodic effect by acting directly on intestinal smooth muscles. It is very unlikely that the antispasmodic effect of BES is due to antimuscarinic actions, since this herbal drug also inhibited the contractions induced by barium chloride which does not act through a receptor-mediated mechanism. As the inhibitory effect of BSE was exerted at postjunctional level, we investigated the mechanism of the antispasmodic activity of this herbal extract on the contractions induced by acetylcholine.

Contractions of all smooth muscles, including those of gastrointestinal tract, absolutely depend on the presence of Ca2+. Agonists-induced contractions may be related to the release of intracellular Ca2+ from sarcoplasmic stores in addition to the influx, mainly through L-type Ca2+ channels of extracellular Ca2+ (Makhlouf, 1994). Consequently, smooth muscle contraction can be abolished by antispasmodic drugs through the inhibition of Ca2+ entry or release into the cells. In the present study, we have shown that verapamil and nifedipine, two blockers of L-type Ca2+ channels, reduced the inhibitory effect of BSE on acetylcholine-induced contractions suggesting the involvement of such channels in the mode of BSE. The lack of effect of cyclopiazonic acid, a well known potent and specific inhibitor of the sarcoplasmic reticulum Ca2+-ATPase in smooth muscles (Grasa et al., 2004), excludes an action of BSE on the release of Ca2+ from sarcoplasmic stores.

Phosphodiesterase (PDE) enzymes are responsible for the breakdown of cyclic nucleotides (Beavo, 1995). At least seven PDE isoenzyme families exist including PDE IV, which is involved in the regulation of intestinal contractility (Bauer et al., 1991; Tomkinson & Raeburn, 1996). Blockade of PDE IV results in an increase of cAMP which in turn decreases calcium entry into the cell through L-type Ca2+ channels (Kaneda et al., 1997). In the present study, we have shown that the PDE IV inhibitor rolipram did not affect the inhibitory effect of BSE on intestinal motility, thus suggesting that BSE possibly affects L-type Ca2+ channels trough a PDE IV-independent mechanism. An unexpected result of the present study was the ability of the PDE IV inhibitor rolipram, given alone, to potentiate acetylcholine-induced contractions. Indeed, inhibition of phosphodiesterase IV is expected to reduce contractions by increasing the basic amount of the relaxing second messenger cAMP (Barbier & Lefebvre, 1995). The reason for this discrepancy remains to be examined.

Boswellic acids are considered to be the ingredients responsible of the plant anti-inflammatory activity, since these compounds inhibit leukotriene biosynthesis by impairing the lipoxygenase activity (Wildfeuer et al., 1998; Safayhi et al., 2000). In the present study, we evaluated the effect of 3-acetyl-11-keto-β-boswellic acid, one of the most effective compounds among the boswellic acids tested so far in vitro (Sailer et al., 1996; 1997). This compound has been found to attenuate experimental ileitis in rats (Krieglstein et al., 2001). Our findings show that 3-acetyl-11-keto-β-boswellic acid effectively reduced acetylcholine-induced contractions, thus suggesting that this compound may be responsible, at least in part, of the antispasmodic action of BSE. As compounds which modulate intestinal contractility in vitro may affect motility in vivo, and because BSE inhibited acetylcholine-induced contractions both in the guinea-pig ileum and in the mouse, we evaluated the effect of BSE on intestinal transit in the mouse in vivo. Moreover, because B. serrata has been clinically evaluated in patients with inflammatory bowel disease, we also studied the effect of these doses of herbal extract in a model of intestinal inflammation. Croton oil is a well known irritant used experimentally to induce inflammation in the mouse small intestine (Pol & Puig, 1997; Izzo et al., 2001). This inflammation is characterized by disruption of the mucosa and an infiltration of lymphocytes into the submucosa associated with an increase of intestinal transit (Pol & Puig, 1997). We found that BSE (in the dose range of 100–400 mg kg−1) was without effect in control animals, but inhibited motility in animals treated with croton oil. Based on human pharmacokinetic data (Mack, 1990) and by assuming similar pharmacokinetics in mice, an oral dose of 100–400 mg kg−1 of BSE should result approximately in plasma concentrations of boswellic acids within the same range as the concentrations tested in vitro. Interestingly, Shi & Sarna (2000) found that Ca2+ influx through L-type Ca2+ channels, but not the release of intracellular Ca2+, is involved in motility changes associated to intestinal inflammation (induced by exposure to ethanol and acetic acid), which is consistent with the ability of verapamil and nifedipine to reduce the inhibitory effect of BSE on motility in vitro (see above). Others have shown that B. serrata resin attenuated macroscopic and microscopic inflammatory features associated with indomethacin-induced ileitis in rats (Krieglstein et al., 2001). By contrast, it has been recently reported that B. serrata did not ameliorate symptoms of dextran sulfate- or trinitrobenzene sulfonic acid-induced colitis in mice (Kiela et al., 2005).

It is well known that drugs which inhibit intestinal transit in pathophysiological state may be effective in alleviating diarrhoea. In addition, because diarrhoea is a major pathophysiological feature in patients with inflammatory bowel disease, we evaluated the potential antidiarrhoeal effect of BSE. We used the castor oil test, which is extensively employed as a basic pharmacological test to screen antidiarrhoeal drugs. One of the assets of this model is the very reproducible evacuation of unformed stools 2-h after laxative administration. We found that BSE reduced castor oil-induced diarrhoea, a relevant finding in the light of the fact that BSE administration is not associated with constipating effects under physiological conditions (see results on intestinal transit described above). Indeed, one of the major side effects associated with oral administration of opiates (the most known antidiarrhoeal agents) is their constipating effect (Jafri & Pasricha, 2001). It is noteworthy that an involvement of L-type Ca2+ channels in the mode of action of BSE is consistent with the ability of verapamil to reduce diarrhoea during small intestinal inflammation (Lee et al., 1997). The effect of BSE could be either due to a direct effect on L-type channel activity or to an indirect effect on cellular membrane which reduce the activity of these channels (e.g. a change of the membrane potential). As a result of its action on L-type calcium channels, BSE could influence cardiovascular functions, including changes in blood pressure. However, BSE does not cause cardiovascular side effects when used in humans (Gupta et al., 1997).

Finally, we investigate the possible role of the lipoxygenase enzyme in the motility changes associated to BSE. Previous investigators have reported that boswellic acids are inhibitors of lipoxygenase enzyme. Moreover, leukotrienes may affect intestinal motility and have been involved in diarrhoeal diseases (Shahbazian et al., 2002). We have shown that the lipoxygenase inhibitor zileuton did not modify the antispasmodic and the antidiarrhoeal effect of BSE, thus suggesting a lack of involvement of leukotrienes in the mode of action of BSE.

In conclusion, the present results demonstrate that BSE can normalize the intestinal motility altered by an inflammatory stimulus and possesses antidiarrhoeal activity. In vitro studies have shown that the extract directly inhibits intestinal motility with a mechanism involving L-type Ca2+ channels. These results could explain, at least in part, the clinical efficacy of this Ayurvedic remedy in reducing diarrhoea in patients with inflammatory bowel disease. Moreover, BSE prevented experimental diarrhoea without slowing the rate of transit, which is of potential clinical interest since currently used antidiarrhoeal agents are often associated with constipating effects.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported by Italian MIUR.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • AMMON, H.P. (1996). Salai Guggal –Boswellia serrata: from a herbal medicine to a non-redox inhibitor of leukotriene biosynthesis. Eur. J. Med. Res., 24, 369370.
  • AMMON, H.P., MACK, T., SINGH, G.B. & SAFAYHI, H. (1991). Inhibition of leukotriene B4 formation in rat peritoneal neutrophils by an ethanolic extract of the gum resin exudate of Boswellia serrata. Planta Med., 57, 203207.
  • AMMON, H.P., SAFAYHI, H., MACK, T. & SABIERAJ, J. (1993). Mechanism of antiinflammatory actions of curcumine and boswellic acids. J. Ethnopharmacol., 38, 113119.
  • ARONSSON, U. & HOLMGREN, S. (2000). Muscarinic M3-like receptors, cyclic AMP and L-type calcium channels are involved in the contractile response to cholinergic agents in gut smooth muscle of the rainbow trout Oncorhynchus mykiss. Fish Physiol. Biochem., 23, 353361.
  • BARBIER, A.J. & LEFEBVRE, R.A. (1995). Relaxant influence of phosphodiesterase inhibitors in the cat gastric fundus. Eur. J. Pharmacol., 276, 4147.
  • BAUER, V., HOLZER, P. & ITO, Y. (1991). Role of extra- and intracellular calcium in the contractile action of agonists in the guinea-pig ileum. Naunyn Schmiedebergs Arch. Pharmacol., 343, 5864.
  • BEAVO, J.A. (1995). Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol. Rev., 75, 725748.
  • BROCCARDO, M., IMPROTA, G. & TABACCO, A. (1998). Central effect of SNC 80, a selective and systemically active delta-opioid receptor agonist, on gastrointestinal propulsion in the mouse. Eur. J. Pharmacol., 26, 247251.
  • CAPASSO, F., GAGINELLA, T.S., GRANDOLINI, G. & IZZO, A.A. (2003). Phytotherapy. A Quick Reference to Herbal Medicine. Springer-Verlag Berlin Heidelberg, Germany.
  • CAPASSO, F., MASCOLO, N., IZZO, A.A. & GAGINELLA, T.S. (1994). Dissociation of castor oil-induced diarrhoea and intestinal mucosal injury in rat: effect of NG-nitro-L-arginine methyl ester. Br. J. Pharmacol., 113, 11271130.
  • CAPASSO, R., IZZO, A.A., FEZZA, F., PINTO, A., CAPASSO, F., MASCOLO, N. & DI MARZO, V. (2001). Inhibitory effect of palmitoylethanolamide on gastrointestinal motility in mice. Br. J. Pharmacol., 134, 945950.
  • GERHARDT, H., SEIFERT, F., BUVARI, P., VOGELSANG, H. & REPGES, R. (2001). Therapy of active Crohn disease with Boswellia serrata extract H 15. Z. Gastroenterol., 39, 1117.
  • GRASA, L., REBOLLAR, E., ARRUEBO, M.P., PLAZA, M.A. & MURILLO, M.D. (2004). The role of Ca2+ in the contractility of rabbit small intestine in vitro. J. Physiol. Pharmacol., 55, 639650.
  • GUPTA, I., PARIHAR, A., MALHOTRA, P., GUPTA, S., LUDTKE, R., SAFAYHI, H. & AMMON, H.P. (2001). Effects of gum resin of Boswellia serrata in patients with chronic colitis. Planta Med., 67, 391395.
  • GUPTA, I., PARIHAR, A., MALHOTRA, P., SINGH, G.B., LUDTKE, R., SAFAYHI, H. & AMMON, H.P. (1997). Effects of Boswellia serrata gum resin in patients with ulcerative colitis. Eur. J. Med. Res., 2, 3743.
  • GUPTA, O.P., SHARMA, N. & CHAND, D. (1992). A sensitive and relevant model for evaluating anti-inflammatory activity-papaya latex-induced rat paw inflammation. J. Pharmacol. Toxicol. Methods, 28, 1519.
  • IZZO, A.A., BORRELLI, F., CAPASSO, F., CAPASSO, R., PINTO, L., CRISTONI, A. & MASCOLO, N. (1999). Contractile effect of (+)-glaucine in the isolated guinea-pig ileum. Eur. J. Pharmacol., 21, 215218.
  • IZZO, A.A., FEZZA, F., CAPASSO, R., BISOGNO, T., PINTO, L., IUOVONE, T., ESPOSITO, G., MASCOLO, N., DI MARZO, V. & CAPASSO, F. (2001). Cannabinoid CB1-receptor mediated regulation of gastrointestinal motility in mice in a model of intestinal inflammation. Br. J. Pharmacol., 134, 563570.
  • IZZO, A.A., PINTO, L., BORRELLI, F., CAPASSO, R., MASCOLO, N. & CAPASSO, F. (2000). Central and peripheral cannabinoid modulation of gastrointestinal transit in physiological states or during the diarrhoea induced by croton oil. Br. J. Pharmacol., 129, 16271632.
  • JAFRI, S. & PASRICHA, P.J. (2001). Agents used for diarrhoea, constipation, and inflammatory bowel disease; agents used for biliary and pancreatic disease. In: GOODMAN & GILMAN'S. The Pharmacological Basis of Therapeutics. eds. Hardman, J.G. & Limbird, L.E.,10th edn., pp. 10371058. New York: McGraw-Hill.
  • KANEDA, T., SHIMIZU, K., NAKAJYO, S. & URAKAWA, N. (1997). Effects of various selective phosphodiesterase inhibitors on muscle contractility in guinea pig ileal longitudinal smooth muscle. Jpn. J. Pharmacol., 75, 7785.
  • KIELA, P.R., MIDURA, A.J., KUSCUOGLU, N., JOLAD, S.D., SOLYOM, A.M., BESSELSEN, D.G., TIMMERMANN, B.N. & GHISHAN, F.K. (2005). Effects of Boswellia serrata in mouse models of chemically induced colitis. Am. J. Physiol. Gastrointest. Liver. Physiol., 288, 798808.
  • KRIEGLSTEIN, C.F., ANTHONI, C., RIJCKEN, E.J., LAUKOTTER, M., SPIEGEL, H.U., BODEN, S.E., SCHWEIZER, S., SAFAYHI, H., SENNINGER, N. & SCHURMANN, G. (2001). Acetyl-11-keto-β-boswellic acid, a constituent of a herbal medicine from Boswellia serrata resin, attenuates experimental ileitis. Int. J. Colorectal. Dis., 16, 8895.
  • LEE, C.W., SARNA, S.K., SINGARAM, C. & CASPER, M.A. (1997). Ca2+ channel blockade by verapamil inhibits GMCs and diarrhea during small intestinal inflammation. Am. J. Physiol., 273, 785794.
  • LIS-BALCHIN, M. & HART, S.L. (2002). Coleonema album: studies of the pharmacological action on smooth muscle in vitro and antimicrobial action of its essential oil. Phytother. Res., 16, 292294.
  • MACK, T.H. (1990). Hemmung der Biosynthese der 5-Lipoxygenaseprodukte stimulierter neutrophiler Granulozyten der Ratte durch Extrakte aus dem Harz von Boswellia serrata Roxbund durch Boswelliasäuren. Doctoral thesis, University of Tuebingen.
  • MAKHLOUF, M.G. (1994). Neuromuscular function of the small intestine. In: Physiology of the Gastrointestinal Tract. ed. L.R. Johnson, 3rd edn., pp. 977990. New York: Raven Press.
  • PINTO, L., IZZO, A.A., CASCIO, M.G., BISOGNO, T., HOSPODAR-SCOTT, K., BROWN, D.R., MASCOLO, N., DI MARZO, V. & CAPASSO, F. (2002). Endocannabinoids as physiological regulators of colonic propulsion in mice. Gastroenterology, 123, 227234.
  • POL, O. & PUIG, M.M. (1997). Reversal of tolerance to the antitransit effects of morphine during acute intestinal inflammation in mice. Br. J. Pharmacol., 122, 12161222.
  • SAFAYHI, H., BODEN, S.E., SCHWEIZER, S. & AMMON, H.P. (2000). Concentration-dependent potentiating and inhibitory effects of Boswellia extracts on 5-lipoxygenase product formation in stimulated PMNL. Planta Med., 66, 110113.
  • SAFAYHI, H., MACK, T., SABIERAJ, J., ANAZODO, M.I., SUBRAMANIAN, L.R. & AMMON, H.P. (1992). Boswellic acids: novel, specific, nonredox inhibitors of 5-lipoxygenase. J. Pharmacol. Exp. Ther., 261, 11431146.
  • SAILER, E.R., HOERNLEIN, R.F., SUBRAMANIAN, L.R., AMMON, H.T.P. & SAFAYHI, H. (1997). Preparation of novel analogues of the nonredox-type non-competitive leukotriene biosyntesis inhibitor AKBA. Arch. Pharm., 329, 5455.
  • SAILER, E.R., SUBRAMANIAN, L.R., RALL, B., HOERNLEIN, R.F., AMMON, H.P. & SAFAYHI, H. (1996). Acetyl-11-keto-β-boswellic acid (AKBA): structure requirements for binding and 5-lipoxygenase inhibitory activity. Br. J. Pharmacol., 117, 615618.
  • SHAHBAZIAN, A., HEINEMANN, A., PESKAR, B.A. & HOLZER, P. (2002). Differential peristaltic motor effects of prostanoid (DP, EP, IP, TP) and leukotriene receptor agonists in the guinea-pig isolated small intestine. Br. J. Pharmacol., 137, 10471054.
  • SHARMA, M.L., KHAJURIA, A., KAUL, A., SINGH, S., SINGH, G.B. & ATAL, C.K. (1988). Effect of salai guggal ex-Boswellia serrata on cellular and humoral immune responses and leucocyte migration. Agents Act., 24, 161164.
  • SHI, X.Z. & SARNA, S.K. (2000). Impairment of Ca(2+) mobilization in circular muscle cells of the inflamed colon. Am. J. Physiol., 278, 234242.
  • SINGH, G.B. & ATAL, C.K. (1986). Pharmacology of an extract of salai guggal ex-Boswellia serrata, a new non-steroidal anti-inflammatory agent. Agents Act., 18, 407412.
  • TOMKINSON, A. & RAEBURN, D. (1996). The effect of isoenzyme-selective PDE inhibitors on methacholine-induced contraction of guinea-pig and rat ileum. Br. J. Pharmacol., 118, 21312139.
  • UYAMA, T., IMAIZUMI, Y. & WATANABE, M. (1992). Effects of cyclopiazonic acid, a novel Ca (2+)-ATPase inhibitor, on contractile responses in skinned ileal ideal smooth muscle. Br. J. Pharmacol., 106, 208214.
  • WILDFEUER, A., NEU, I.S., SAFAYHI, H., METZGER, G., WEHRMANN, M., VOGEL, U. & AMMON, H.P. (1998). Effects of boswellic acids extracted from a herbal medicine on the biosynthesis of leukotrienes and the course of experimental autoimmune encephalomyelitis. Arzneimittelforschung, 48, 668674.