Possible Mechanism for the Relaxant Effect of Pimpinella anisum. on Guinea Pig Tracheal Chains

ABSTRACT

Pimpinella anisum. L. is a grassy plant that is believed to have several therapeutic effects including antiasthmatic uses. In a previous study, the relaxant (bronchodilatory) effect of this plant on isolated guinea pig tracheal chains was demonstrated. To study mechanisms responsible for this effect, the inhibitory effect of this plant on contracted tracheal chains of guinea pig was evaluated in the current study. The relaxant effects of aqueous and ethanol extracts and essential oil from P. anisum. compared to negative controls (saline for aqueous extract and essential oil and ethanol for ethanol extract) and positive control (diltiazem) were examined on precontracted isolated tracheal chains of the guinea pig by 10 µM methacholine (group 1) and 60 mM KCl (group 2, n = 7 for each group). In group 1 experiments, diltiazem, both extracts, and essential oil from P. anisum. showed significant relaxant effect on methacholine-induced contraction of tracheal chains compared to those of negative controls (p < 0.05 to p < 0.001). In addition, the effects of extracts were significantly greater than that of diltiazem (for aqueous and ethanol extracts p < 0.05 and p < 0.01, respectively). However, in group 2 experiments, only diltiazem and ethanol extract showed significant relaxant effects on KCl-induced contraction of tracheal chains (p < 0.05 for ethanol extract and p < 0.001 for diltiazem). Relaxant effects of extracts and essential oil obtained in group 2 experiments were significantly lower than those of group 1 (p < 0.05 to p < 0.001). These results confirm the bronchodilatory effects of extracts and essential oil from P. anisum.. However, with regard to the effect of KCl on calcium channels, the results indicated that inhibitory effect of only ethanol extract from P. anisum. on calcium channels may contribute to its relaxant (bronchodilatory) effects on guinea pig tracheal chains. In addition, the results suggest a potassium channel opening effect for this plant, which may contribute on its relaxant effect on tracheal chains of guinea pig.

Keywords:

• Guinea pig tracheal chains
• Pimpinella anisum
• relaxant effect
• possible mechanism(s)

Introduction

Pimpinella anisum. L. (Umbelliferae) is a grassy plant with white flowers and small green to yellow seeds that grows in Egypt, Turkey, and Greece. The seeds of Pimpinella anisum. contain anethole (Chandler & Hawkes, 1984), pseudoisoeugenol (Reichling et al., 1995), coumarins, scopoletin, umbelliferon, estrols (Burkhardt et al., 1986), terpene hydrocarbons (Kartnig et al., 1975), polyenes and polyactylenes (Schulte et al., 1970).

Several therapeutic effects, including for digestive disorders, and for gynecologic, anticonvulsant, antiasthma, and dyspnea purposes, have been described for the seeds of P. anisum. in Iranian ancient medical books (Aboabrahim, 1970). There is evidence of relaxant effects of volatile oil from this plant on isolated tracheal muscles of guinea pigs (Reiter & Brandt, 1985). However, the contractile effect of anethole, the major constituent of this plant, on ileal smooth muscle of guinea pigs has also been demonstrated (Reiter & Brandt, 1985). In contrast, it has been shown that an-ethole has a relaxant effect on skeletal muscle (Albuquerque et al., 1995). In a previous study, we have demonstrated relaxant effects of different extracts and essential oil from P. anisum. on guinea pig tracheal chains (Boskabady & Ramazani-Asari, 2001).

In the current study, other possible mechanisms for the relaxant effect of essential oil, aqueous, and ethanol extracts of this plant on guinea pig tracheal chains were studied.

Materials and Methods

Plant and extracts

Botanists in the herbarium of Ferdowsi University, Mashhad, identified Pimpinella anisum.; the specimen number of the plant is 7701. The plant extracts were prepared as follows: For aqueous extract, 50 g of the chopped, dried plant were extracted with 300 ml distilled water with a Soxhlet apparatus. Ethanol extract was prepared the same as aqueous extract except the solvent was ethanol instead of distilled water. The solvent of both extracts was then removed under reduced pressure until the extract volume reached 20 ml. Plant ingredient concentration in the final extracts was 33.3% w/v in both aqueous and ethanol extracts. From 100 g of the chopped, dried plant with 1000 ml distilled water, 1 ml essential oil was extracted by steam-distilled apparatus. The concentration of plant ingredients in essential oil was 10% v/v.

Tissue preparations

Male guinea pigs (400–700 g) were killed by a blow on the neck, and tracheas were removed. Each trachea was cut into 10 rings (each containing 2–3 cartilaginous rings). All the rings were then cut open opposite the trachealis muscle and sutured together to form a tracheal chain (Holroyde, 1986). Tissue was then suspended in a 10-ml organ bath (organ bath 61300, BioScience Palmer-Washington, Sheerness, Kent, UK) containing Krebs-Henseliet solution of the following composition (mM): NaCl 120, NaHCO₃ 25, MgSO₄ 0.5, KH₂PO₄ 1.2, KCl 4.72, CaCl₂ 2.5, and dextrose 11. The Krebs solution was maintained at 37°C and gassed with 95% O₂ and 5% CO₂. Tissue was suspended under isotonic tension of 1 g and allowed to equilibrate for at least 1 h while it was washed with Krebs solution every 15 min.

Protocols

The relaxant effects of 0.02 ml essential oil, 0.6 ml of aqueous and 0.1 ml ethanol extracts from P. anisum. and 5 μm diltiazem as positive control (Sigma Chemical Ltd, Poole Dorset, UK) on calcium channels of isolated guinea pig tracheal chains were examined. Saline (0.6 ml) was used as negative control for essential oil and aqueous extract and ethanol (0.1 ml) for ethanol extract.

In each experiment, the effect of one of the solutions on contracted tracheal smooth muscle was measured after exposing tissue to the solution for 10 min. A decrease in tone was considered as a relaxant effect and expressed as positive percentage change, and an increase in tone was considered as a contractile effect and expressed as negative percentage change in proportion to maximum contraction obtained due to contractile agents (Martin et al., 1994). The relaxant effect of different solutions was tested with two different experimental designs as follows:

1. On tracheal chains contracted by 10 µM methacholine hydrochloride (Sigma Chemical Ltd UK) (group 1 experiments).

2. On tracheal chains contracted by 60 mM KCl (group 2 experiments).

The relaxant effects in two groups of experiments were examined in two different series of tracheal chains (for all groups, n = 7). All of the experiments were performed randomly with a 1-h resting period of tracheal chains between each two experiments while washing the tissues every 15 min with Krebs solution. In all experiments, responses were recorded on a kymograph (ET8 G-Boulitt, Paris, France) and after fixation were measured.

Statistical analysis

The data of the relaxant effect of different experiments were expressed as mean ± SEM. The data of relaxant effects of different concentrations of extracts and diltiazem were compared using the ANOVA test. Significance was accepted at p < 0.05.

Results

In group 1 experiments, diltiazem, both extracts, and essential oil from P. anisum. showed significant relaxant effect on methacholine-induced contractions of guinea pig tracheal chains (p < 0.05 to p < 0.001). The effects of both extracts were significantly greater (for aqueous and ethanol extracts, p < 0.05 and p < 0.01, respectively) than that of diltiazem (Table 1; Fig. 1a).

Table 1.. Relaxant effect of extracts and essential oil from Pimpinella anisum. on contracted trachealrings of guinea pig in comparison to negative control (ethanol for ethanol extract, saline for aqueous extract and essential oil) and positive control (diltiazem) in group 1 (contracted tissues by 10 µM methacholine) and group 2 experiments (contracted tissues by 60 mM KCl) as percentage response relative to maximum contraction due to contractile agents.

Experimental designs	Group 1	Group 2
Saline	0.17 ± 0.12	−5.18 ± 3.42
Ethanol	23.71 ± 5.19	−8.7 ± 4.95
Aqueous extract	41.31 ± 10.41 ***^†	−7.27 ± 7.54 NS, ††^‡‡
Ethanol extract	70.86 ± 5.49 **^††	10.01 ± 8.33 ^†† ^‡‡‡
Essential oil	20.5 ± 5.34 ^,ns	−8.35 ± 13.95 NS, ††^‡
Diltiazem	19.66 ± 5.07	90.71 ± 6.21 ***^‡‡‡

Values are presented as mean ± SEM.

Statistical differences between the effects of extracts, essential oil, and positive controls and those of negative control: NS, non-significant difference; *p < 0.05; **p < 0.01; ***p < 0.001.

Statistical differences between the effects of extracts and essential oil and those of positive control: ns non-significant difference; †p < 0.05; ††p < 0.01.

Statistical differences between the result of two groups; ‡p < 0.05; ‡‡p < 0.01; ‡‡‡p < 0.001.

In group 2 experiments, only the ethanol extract from P. anisum. showed a significant relaxant effect on KCl-induced contraction of guinea pig tracheal chains (p < 0.05). In fact, the aqueous extract and essential oil showed nonsignificant contractile effect compared to that of saline (Table 1; Fig. 1b). The effects of extracts and essential oil were significantly different with that of diltiazem (p < 0.01 for all cases; Table 1; Fig. 1b).

Figure 1. Relaxant effects of different extracts and essential oil from Pimpinella anisum. and saline, ethanol, and diltiazem on contracted tracheal rings of guinea pig tracheal chains in (a) group 1 (contracted tissues by 10 µM methacholine and (b) group 2 experiments (contracted tissues by 60 mM KCl) (for each group, n = 7). Differences between relaxant effect of negative controls (ethanol for ethanol extract and saline for aqueous extract and essential oil) with the effect of plant solutions: NS, non-significant difference; *p < 0.05; **p < 0.01; ***p < 0.001. Statistical differences between the effects of extracts and essential oil with those of positive control: ns, non-significant difference; †p < 0.05; ††p < 0.01.

The effects of extracts and essential oil in group 2 experiments were significantly lower than those of group 1 (p < 0.05 to p < 0.001, Table 1). However, the effect of diltiazem in group 2 was significantly greater than that of group 1 (p < 0.001; Table 1).

Discussion

In the current study, alternative mechanisms for the relaxant effects of essential oil and extracts (aqueous and ethanol) of P. anisum. on tracheal chains of guinea pig were studied. In group 1 experiments, essential oil, ethanol extract, and diltiazem showed significant relaxant effects on methacholine-induced contraction of tracheal chains. The results of this group support the findings of previous studies demonstrating relaxant effect of volatile oil from P. anisum. on isolated tracheal and ileal smooth muscle of the guinea pig (Reiter & Brandt, 1985) and relaxant effect of essential oil and extracts from this plant on tracheal chains (Boskabady & Ramazani-Asari, 2001). The relaxant effects of both extracts were significant, and the effect of essential oil was not significantly greater than that of diltiazem in this group of study.

In group 2, only the ethanol extract from P. anisum. showed a relaxant effect on KCL-induced contractions of tracheal chains. However, the relaxant effects of both extract and essential oil were significantly lower than that of diltiazem in group 2 experiments.

While KCl affects calcium channels (Perez-Guerrero et al., 1997) and, with regard to bronchodilatory effect of calcium channel blockers (McCaig & DeJonckeere, 1993; Miyahara et al., 1993), these findings showed the absence of a blocking effect of aqueous extract and essential oil from P. anisum. on calcium channels. The absence of obvious relaxant effect of aqueous extract and essential oil from this plant in group 2 and the relatively potent relaxant effect of aqueous extract and essential oil in group 1 experiments may also indicate an opening effect of these fractions on potassium channels, because the bronchodilatory effect of potassium channel opening has been demonstrated previously (Buckle et al., 1993). If the aqueous extract and essential oil from P. anisum. have a potassium channel opening effect, they would not have a relaxant effect on tracheal chains contracted by KCl, while they could show a relaxant effect when the tracheal chain was contracted by methacholine. However, the results of the current study suggest an inhibitory effect of ethanol extract on calcium channels.

The lower relaxant effect observed for essential oil compared to those of two extracts in group 1 experiments could be due to lower volume of essential oil, existence of greater than ethanol, which has a contractile effect on smooth muscle (Reiter & Brandt, 1985), or lower effective substance(s) in essential oil than extracts. This is presumably due to the variation of methods of extraction between extracts and essential oil. The most probable explanation for this finding could be the existence of anethol in the essential oil of this plant, because the effective substance is usually greater in essential oil than in other extracts of plants. Indeed, in previous studies, the greater relaxant effect for essential oil than those of other extracts for some plants on guinea pig tracheal chains have been observed (Boskabady et al., 1998; Boskabady & Talebi, 1999). The relaxant effect of ethanol may suggest that the effect of ethanol extract might be due to its solvent content. However, the relaxant effect of ethanol extract was significantly higher than the same volume of ethanol. In addition, in group 2 experiments, ethanol did not show any relaxant effect on tracheal chains while ethanol extract caused a significant relaxant effect.

Our previous study also showed that histamine (H¹) receptor inhibitory and β-adrenoceptor stimulatory effects of this plant do not contribute to the relaxant properties of the plant. However, inhibitory properties of the plant at muscarinic receptors (anticholinergic effect) may, at least in part, be responsible for this broncodilatory effect.

The other possible mechanisms causing bronchodilatory effect include stimulation of inhibitory nonadrenergic, noncholinergic nervous system (NANC) or inhibition of stimulatory NANC (Linden et al., 1993), methyl xanthine activity (Meini et al., 1993), and inhibition of phosphodiesterase (Van Amsterdam et al., 1989). The contributions of these mechanisms on the bronchodilatory effect of P. anisum. should be clarified in further studies.

In conclusion, the results of the current study confirmed the relaxant effect of essential oil and extracts from P. anisum.. However, the relaxant effects of the aqueous extract and essential oil from this plant are not due to the inhibitory effect of the plant on calcium channels. The results suggest a potassium channel opening effect for this plant, which may contribute to its relaxant effect on tracheal chains of guinea pig. In addition, the results suggest a calcium channel inhibitory effect for ethanol extract.

Acknowledgment

The authors would like to thank Dr. M. Ramazani for his assistance in preparation of the extracts.