Relaxant effect of ethanol extract of Carum carvi on dispersed intestinal smooth muscle cells of the guinea pig

Abstract

The present study investigates the direct effects of Carum carvi L. (Apiaceae) ethanol extract on dispersed intestinal smooth muscle cells (SMC) of guinea pigs. Effects of the plant extract on SMC and of acetylcholine (Ach) on extract pretreated SMC were measured by micrometric scanning technique. Three different extract concentrations (2.5 mg/mL, 250 μg/mL, and 25 μg/mL) were used. Ethanol extract of C. carvi reduced significantly the response of dispersed SMC to Ach. Pretreatment of SMC with the highest concentration of C. carvi ethanol extract (2.5 mg/mL) has significantly inhibited the response of SMC to Ach. The data obtained indicate a dose-dependent inhibition of the contraction induced by Ach. This response may explain, in part, the beneficial effect of caraway in relieving gastrointestinal symptoms associated with dyspepsia.

Keywords::

• Carum carvi
• intestine
• smooth muscle
• relaxation
• guinea pig

Introduction

Carum carvi L. (Apiaceae), rich in fixed oils, proteins, and polysaccharides, is common in the Middle East (Bruneton, 1999; Evans, 2002). Volatile oils (carvone and limonene as main volatile principles) and furanocoumarins (bergaptene and xanthotoxine) are reported as the main active ingredients (Blumenthal et al., 1998; Heinrich et al., 2004; Jaenicke et al., 2003). Aromatic glucosides and glucides were also detected as water soluble constituents in caraway (Matsumara et al., 2002).

In Jordan caraway is commonly used as a condiment and a home remedy to treat different gastro-intestinal and respiratory problems (Abu-Irmaileh & Afifi, 2000). Among its reported activities are carminative, spasmolytic, expectorant, nerve calmative, digestive, emmenogogue, antimicrobial and antirheumatic. It is also used externally as a rubefacient and in the cosmetic industry (Boulos, 1983; Blumenthal et al., 1998; Heinrich et al., 2004; Jaenicke et al., 2003).

Carvone and limonene, isolated from caraway oil, were found to induce the detoxifying enzyme glutathione-S-transferase in several mouse target tissues (Zheng et al., 1992). Carvone, specifically, was found to be responsible for the high enzyme-inducing and related anticarcinogenic activities. A dose-dependent antiulcerogenic activity, associated with a reduced acid output and an increased mucin secretion as well as an increase in prostaglandin E₂ release and a decrease in leucotrienes, was reported by Khayyal et al. (2001). Moreover, antibacterial and potent antioxidant activities (through hydroxyl and lipid peroxide superoxide radicals inhibition) have been demonstrated for C. carvi (Iacobellis et al., 2005; Satyanarayana et al., 2004).

In clinical studies, phytotherapeutic combinations containing caraway oil exhibited beneficial effects on gastro-intestinal symptoms, such as dyspepsia and functional dyspeptic syndrome (Coon & Ernst, 2002; Freise & Kohler, 1999; Madisch et al., 1999, 2004; Mascher et al., 2001; May et al., 1996, 2000; Micklefield et al., 2003). None of these studies clearly defined the effects of caraway on gastro-intestinal motor activities, as the clinical dyspeptic syndrome is a complex pathophysiological process in which alteration of motility, alteration of sensation as well as psychosocial factors are involved in the generation of dyspepsia (Camilleri, 2001). Caraway oil inhibited the motor activities of SMC of the gallbladder, stomach, trachea and ileum (Boskabady et al., 2003; Goerg & Spilker, 2003; Micklefield et al., 2003; Reiter & Brandt, 1985). Recently, in vitro and in vivo experiments demonstrated antihypertensive, antispasmodic, bronchodilator and hepatoprotective activities for C. copticum seeds (Gilani et al., 2005).

The present study investigated the direct effect of C. carvi ethanol extract on SMC contractions. The enteric nervous system is involved in the regulation of motor functions in the gastro-intestinal tract. This, in turn, might mask the plant extract’s direct effect on SMC. Therefore, cell dispersion was used as a measure to overcome this phenomenon.

Materials and methods

Plant material

Commercially available caraway fruits were purchased in Amman and authenticated by one of the authors (F.U. Afifi). A voucher specimen has been deposited in the Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan.

Preparation of the extract

Coarsely powdered dry caraway fruits (100 g) were refluxed with 1 L of 96% ethanol. The extract was kept overnight at room temperature and used after filtration for thin layer chromatographic (TLC) screening. Carvone and limonene were identified based on their Rf-values and color reactions and in comparison to the reference substances as described by Wagner and Bladt (1996). For in vitro experiments the alcoholic extract was evaporated until complete dryness and the syrupy residue was dissolved in distilled water (2.5 g/10 mL). This stock solution was used for the preparation of the final concentrations in the test tubes.

Animals

Locally inbred guinea pigs of both sexes weighing 350-450 g were included in the study. The animals were kept in the animal house unit of the Faculty of Medicine at the University of Jordan for two weeks to be acclimatized. They were provided with standard pellet laboratory diet and water ad libitum. All animal experiments were conducted in concordance with the University of Jordan’s “Regulations and Ethical Guidelines for the Care and Use of Laboratory Animals”. The funding for the project was granted upon providing proof for compliance with these regulations.

Dispersion of smooth muscle cells

On the day of the experiment, fully anesthetized guinea pigs were sacrificed by opening of the chest cavity. The whole small intestine was removed and placed in freshly prepared Krebs solution (pH 7.4), previously aerated with 95% O₂ and 5% CO₂. The small intestine was cut into small pieces of 2-2.5 cm. After removal of the longitudinal layer and mucosa by mechanical scraping, the circular layer was taken, and prepared for enzymatic digestion by chopping. SMC were dispersed by enzymatic digestion as described by Murthy et al. (1991). Briefly, chopped preparation was placed into 30 mL Krebs solution containing 320 U collagenase/mL.The digestion process continued for about 90 min at 35°C with very low bubbling by 95% O₂ and 5% CO₂. An aliquot containing dispersed SMC after the process of digestion was filtered through nitex mesh of 500 μm. The filtrate was centrifuged for 10 min at 3000 rpm. After this process the digestive solution was removed and cells were resuspended in collagenase-free Krebs solution for washing and were centrifuged twice. After the second wash, cells were suspended into Krebs solution and were ready to be used in the present study.

Experimental protocol

Equal volumes of solution containing 900 μL of dispersed cells were transferred into test tubes. The tubes were divided into a control group and an extract treated group. The control group included one blank tube containing only dispersed cells (C) and a second one containing the acetylcholine (Ach) added dispersed cells (CAch) to give a final concentration of 5 × 10⁻⁵ M. In this group, muscle activity was stopped 15 sec after Ach-treatment using 1% acrolein (Aldrich, Germany). The extract-treated groups comprised those treated only with extract (E) and the Ach added group (EAch) after pre-treatment with the extract for 45 sec. The final concentrations of the extract in the solutions with the dispersed cells were 2.5 mg/mL, 250 μg/mL, and 25 μg/mL. In both groups, 15 sec after Ach addition to EAch, the muscle activity was stopped by addition of 1% acrolein. For micrometric scanning, dispersed cells were spread over microscope slides, covered with cover glass and kept overnight in a humid atmosphere at 4°C prior to the measurement (Bitar et al., 1979; Murthy et al., 1991).

Measurement of dispersed SMC

On the second day, the length of SMC was measured by micrometric scanning technique as described by Murthy et al. (1991). The mean length of the first 50 encountered cells was taken from each slide and considered as one experimental result.

Analysis of results

All data are presented as means of smooth muscle length ± SEM. The differences between means of the control (Blank and Ach treated control) were compared to extract-treated and extract-pretreated samples by using Student’s t-test. Significance was considered when p value is <0.05.

Results

Ethanol extract of caraway reduced significantly the response of dispersed SMC from the circular layer of guinea pig small intestine to Ach. In the control tubes (C), the mean length of SMC from the small intestine was 105.8 ± 1.3 μm (n = 9) (Figure 1). After inducing contraction with Ach (5 x 10⁻⁵M), the mean length of SMC was 69.7 ± 1.3 μm (n = 9) in the CAch-group (Figure 2). This result indicates a 34% decrease in the SMC length following Ach treatment.

Figure 1.  The effect of C. carvi extract on dispersed small intestinal SMC. Results are means ± SEM of 6-9 experiments. Significance of difference from corresponding control value: *p <0.05.

Figure 2.  Effect of Ach on C. carvi extract pretreated SMC. Results are means ±SEM of 6-9 experiments. Significance of difference from corresponding control value: *p <0.05.

Pre-treatment of the cells with extract concentration of 2.5 mg/mL has significantly reduced the contractile response of SMC to Ach. The mean length of SMC in this group was 92.2 ± 1.5 μm (n = 6). Pre-treatment of cells with lower concentrations of the extract has not reduced the contractile response to Ach. In these groups, the mean length of SMC was 71.8 ± 1.7 μm (n = 6) and 66.7 ± 2.9 μm (n = 6) at extract concentrations of 250 μg/mL and 25 μg/mL, respectively (Figure 2).

The effect of the extract on the length of SMC was also studied. Cells treated with the highest concentration (2.5 mg/mL) of the ethanol extract have not shown significant changes in their mean length. In this group, the mean length of cells was 99.3 ± 4.3 μm (n = 6). On the contrary, a significant decrease in the mean length of the SMC to 93 ± 4 μm (n = 6) and 86.6 ± 3.1 μm (n = 7) was observed at concentrations of 250 and 25 μg/mL respectively (Figure 1), which may indicate a slight contractile response when cells were treated with the extract at low concentrations.

Discussion

One of the advantages of studying the effects of test substances on dispersed SMC is the exclusion of the effects mediated by the enteric nervous system. Accordingly, direct effects of test substances on SMC can be observed and evaluated.

In the present study, pretreatment of SMC with the highest concentration of C. carvi ethanol extract has significantly inhibited the response of SMC to Ach. Nevertheless, the contractile responses to Ach have not been changed when cells were pre-treated with lower concentrations of the extract (Figure 2). This indicates a dose-dependent inhibition of SMC response to Ach, since increasing the concentration of the ethanol extract caused almost complete inhibition at the highest dose of the extract used. These findings strongly indicate that the ethanol extract of caraway contains constituents that inhibit the activity of the SMC.

Furthermore, a slight but significant decrease of about 18% in the length of SMC treated with the lowest concentration of the ethanol extract was observed (Figure 1). The absence of contractile effect on SMC in high concentration is probably influenced by some inhibitory substances found in the extract which exert their relaxant activity at higher concentrations.

In previous in vitro studies, reduced activity of tracheal SMC by C. carvi and C. copticum L. extracts was demonstrated (Boskabady et al., 2003; Reiter & Brandt, 1985). Recently, Gilani and his co-workers (2005) demonstrated that C. copticum extract has reduced spontaneous contractile activity and inhibited K⁺-induced contractions in intestinal preparation of rabbits. In clinical trials on healthy volunteers, inhibitory effects of caraway extract on the number of contractions and contraction amplitudes of the migrating motor complex and gall-bladder emptying have been observed (Goerg & Spilker, 2003; Micklefield et al., 2003). These reported findings may support the inhibitory effect of caraway extract on SMC found in the present study.

On the other hand, the inhibitory effect of the caraway oil on the myenteric plexus-longitudinal muscle preparation has not been clearly demonstrated in an in vitro study with caraway oil (Reiter & Brandt, 1985). Similar observation in a clinical trial on healthy volunteers reported no significant changes in gastric emptying and orocecal transit time (Goerg & Spilker, 2003). In these studies the effect of caraway extract on stimulated motor activities was not tested and the interference of enteric neurons is probably responsible for masking the inhibitory effect on the SMC. Abnormal gastro-intestinal motor activities and alteration of visceral sensation are employed in the pathophysiological mechanisms responsible for dyspepsia (Thumshirn, 2002; Timmons et al., 2004). The lack of knowledge about pharmacological effects of extracts of caraway on neural cells and the mode of action on intestinal SMC make it hard to clarify the mechanism by which caraway is reducing functional dyspeptic symptoms. Reduced responses of treated SMC with caraway extract to Ach found in our study may be in part involved in the beneficial effect of caraway in relieving gastrointestinal symptoms associated with dyspepsia. It is not known whether this effect can include also responses of gastro-intestinal SMC to other excitatory stimuli including neurotransmitters released by enteric neurons. The involvement of visceral sensation in the mechanism of dyspepsia and the decrease in pain sensation in dyspeptic patients to phytotherapeutic combination containing caraway extract may indicate also effects on neural cells (Madisch et al., 1999; May et al., 1996; Thumshirn, 2002; Timmons et al., 2004). Future studies should be aimed at investigating the effects on enteric neurons, to clarify the mechanism by which caraway extract has caused relaxant effect on SMC and to identify and test the active materials that caused this effect.

Acknowledgements

The authors are grateful to Jalal Zeidan and Ismail Abaza for technical help.

Declaration of interest: This work was supported by a grant of the Deanship for Scientific Research, University of Jordan. The authors alone are responsible for the content and writing of the paper.