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Inhibitory effect of Bunium persicum on histamine ([H.sub.1]) receptors of guinea pig tracheal chains.

Abstract

The antihistaminic effects of aqueous and macerated extracts, essential oil, 20 nM chlorpheniramine, and saline were tested by performing the cumulative log concentration-response curves of histamine-induced contraction of isolated guinea pig tracheal chains incubated with three different conditions including: (1) 1.4 [micro]M indomethacin, (2) indomethacin, 1 [micro]M propranolol, and 10 nM atropine, and (3) indomethacin and propranolol (for each group n = 8).

The results showed clear parallel rightward shifts in histamine-response curves obtained in the presence of macerated extract in group 2, aqueous extract in group 3, and essential oil in groups 2 and 3 experiments compared with the curves obtained in the presence of saline. The E[C.sub.50] (effective concentration of histamine causing 50% of maximum response) obtained in the presence of essential oil, extracts, and chlorpheniramine in all three sets of experiments were significantly higher than that of saline (P < 0.05 to p < 0.001). The maximum response obtained in the presence of aqueous extract in group 3 compared to group 1 and that of macerated extract in group 2 compared to the other two sets of experiments were improved.

These results indicated a competitive antagonistic effect of Bunium persicum at histamine [H.sub.1] receptors.

[c] 2004 Published by Elsevier GmbH.

Keywords: Bunium persicum; Antihistaminic effect; Trachea; Guinea pig

**********

Introduction

Bunium persicum (Boiss.) B. Fedtsch. (or Carum persicum) is a grassy plant with white or pink flowers and small brownish seeds which grows in warm climate areas of Iran. The seeds of B. persicum contain p-Mentha-1,4-dien-7-al, gamma-terpinene, beta-pinene and cuminaldehyde (Baser et al., 1997).

Several therapeutic effects including those on digestive disorders, urinary tract disorders, and diuretic, gynaecologic, anticonvulsion, antihelmetic and also antiasthma and dyspnoe have been described for the seeds of B. persicum in Iranian ancient medical books (Avesina, 1991).

In previous studies, antimicrobial (Syed and Hanif, 1985) and antifungal (Sardari et al., 1998) effects of this plant have been demonstrated. In our recent study, relaxant and functional antagonistic effects on muscarinic receptors of isolated guinea pig tracheal chains were demonstrated for an extract of Carum carvi a related spezies of Bonum persicum (Boskabady and Talebi, 1999).

To elucidate the other mechanisms responsible for the observed bronchodilatory effect of B. persicum, the inhibitory effect of this plant on histamine [H.sub.1] receptors of guinea pig tracheal chains in comparison with both saline and chlorpheniramine were examined in this study.

Material and methods

Plant and extracts

B. persicum was purchased from local market and identified by botanists in the Herbarium of Ferdowsi University of Mashhad and the specimen number of the plant is 17612. The plant extracts were prepared as follows: For preparing the macerated extract, 50 g of the chopped, dried plant material were macerated with 300 ml distilled water and shaken on a shaker for 48 h. For aqueous extract, the same amount of plant was extracted with 300 ml distilled water in a soxhlet apparatus. The solvent of both extracts was then removed under reduced pressure until the extracts volume reached 20 ml. From 100g of the chopped, dried plant material with 1000 ml distilled water, by steam distilled apparatus 1 ml essential oil was obtained. The plant ingredient concentrations in the final extracts were 45.8% and 36.9% W/W in macerated and aqueous extracts, respectively, and 1.5% V/V in essential oil.

Tissue preparations

Male guinea pigs (400-700 g) were killed by a blow on the neck and trachea removed. Tracheal chains were prepared as previously described (Holroyde, 1986).

Tissue was then suspended in a 10 ml organ bath (organ bath 61300, Bio Science Palmer-Washington, Sheerness, Kent UK) containing Krebs-Henseleit solution of the following composition (mM): NaCl 120, NaHC[O.sub.3] 25, MgS[O.sub.4] 0.5, K[H.sub.2]P[O.sub.4] 1.2, KCl 4.72, Ca[Cl.sub.2] 2.5 and dextrose 11.

The Krebs solution was maintained at 37[degrees]C and gassed with 95% [O.sub.2] and 5% C[O.sub.2]. The tissue was suspended under an 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 inhibitory effect of B. persicum on histamine [H.sub.1] receptors was examined by producing cumulative log concentration-response curve of histamine acid phosphate (BDH Chemical Co, Ltd UK) induced contraction of tracheal chains 10 min after exposing tissue to one solution (essential oil 3 [micro]l, macerated and aqueous extracts 0.08 ml, 0.2 ml of 1 [micro]M chlorpheniramine maleate (Sigma Chemical Ltd UK), or 0.08 ml saline). The consecutive concentrations of histamine were added every 2 min (range 0.1-1000 [micro]M); and the percentage of contraction due to each concentration in proportion to the maximum contraction obtained in the presence of saline was plotted against log concentration of histamine. The effective concentration of histamine causing 50% of maximum response (E[C.sub.50]) in each experiment was measured using the log concentration-response curve of corresponding experiment. The slopes of the histamine-response curve of each experiment were also measured. In experiments with parallel shift in histamine-response curve, the concentration-ratio minus one (CR-1) as competitive antagonism effect was calculated by the following equation:

[[E[C.sub.50] obtained in the presence of effective solutions]/[E[C.sub.50] obtained in the presence of saline]] - 1.

The inhibitory effect of B. persicum on histamine [H.sub.1] receptors was tested on incubated tracheal chains 30 min prior to beginning and during obtaining histamine-response curve with three different experimental designs (for each design, n = 8) as follows:

1. 1.4 [micro]M indomethacin (Sigma Chemical Ltd. UK), (group 1 experiments).

2. 1.4 [micro]M indomethacin, 1 [micro]M propranolol hydrochloride (Sigma Chemical Ltd. UK), and 10 nM atropine sulphate (Sigma Chemical Ltd. UK), (group 2 experiments).

3. 1.4 [micro]M indomethacin and 1 [micro]M propranolol hydrochloride (group 3 experiments).

All of the experiments were performed randomly with 1 h resting period of tracheal chains between 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) and were measured after fixation.

Statistical analysis

All data were expressed as mean[+ or -]SEM. The results (E[C.sub.50], slope of histamine-response curves, and maximum response to histamine) obtained in the presence of essential oil, extracts, and chlorpheniramine were compared with those obtained in the presence of saline and values of (CR-1) obtained in the presence of extracts and essential oil with those of cholorpheniramin using paired "t" test. The results of three groups were compared using one-way analysis of variance test. Significance was accepted at p < 0.05.

Results

Cumulative log concentration-response curves of histamine obtained in the presence of essential oil, extracts, and chlorpheniramine in all three experimental groups showed clear rightward shift compared to histamine-response curves produced in the presence of saline (Fig. 1).

[FIGURE 1 OMITTED]

The E[C.sub.50] of histamine obtained in the presence of essential oil, extracts, and chlorpheniramine in all three experimental conditions were significantly higher than those for saline (p<0.05 to p<0.001). Only E[C.sub.50] obtained in the presence of essential oil and macerated extract in group 3 were significant higher than those of group 1 experiments (p<0.05 for both cases), (Table 1).

The slope of histamine-response curves obtained in the presence of essential oil obtained in groups 2 and 3 (p<0.05 for both cases) and that of aqueous extract in group 3 was significantly higher than group 1 (p<0.001). The slope of histamine-response curve obtained in the presence of macerated extract obtained in group 2 was also significantly higher than those of other two groups (p<0.001 for both cases; Table 2).

The maximum response to histamine obtained in the presence of essential oil and extracts from B. persicum were significantly lower than those of saline in all three sets of experiments (P < 0.01 to P < 0.001). However, the maximum response to histamine obtained in the presence of aqueous extract in group 3 experiments was significantly improved compared to group 1 (p<0.05) and that of macerated extract in group 2 was improved compared to the other two sets of experiments (p < 0.001 vs. group 1 and p < 0.05 vs. group 3), (Table 3).

The values of (CR-1) obtained in the presence of essential oil (1.23[+ or -]0.31) and macerated extract (1.40 [+ or -] 0.28) in group 2 and those of essential oil (1.00 [+ or -] 0.21) and aqueous extract (0.68[+ or -]0.14) in group 3 experiments showed non-significant difference with that of chlorpheniramine (1.06 [+ or -] 0.16 and 1.06 [+ or -] 0.28 for groups 2 and 3 respectively).

Discussion

The bronchodilatory effect seen for B. persicum in our previous study (Boskabady and Talebi, 1999) might be produced due to several different mechanisms. One possible mechanism responsible for this effect could be the inhibitory effect of this plant on histamine [H.sub.1] receptors (Popa, 1977). The inhibitory effect of the essential oil and extracts from this plant was therefore examined on isolated guinea pig tracheal preparations in this study.

The non-parallel rightward shifts in histamine log concentration-response curves, obtained in the presence of essential oil and extracts, greater E[C.sub.50] but lower maximum contraction effect to histamine compared to those of saline in group 1 (incubated trachea with only indomethacin) indicated a functional antagonistic effect of B. persicum at histamine [H.sub.1] receptors of guinea pig trachea (Arunlakshana and Schild, 1959; Ariens, 1987; Linden et al., 1993).

To evaluate the contribution of [beta]-adrenergic stimulatory and/or muscarinic blocking effect on functional antagonism of B. persicum at histamine [H.sub.1] receptors, the antihistaminic effects of extracts and essential oil from this plant were also examined on incubated tracheal preparation with indomethacin, propranolol, and atropine. The parallel rightward shift in histamine-response curves obtained in the presence of macerated extract and essential oil compared to that of saline and significant improvement of maximum responses to histamine obtained in the presence of these two solutions in this group, relative to those of group 1 showed possible competitive antagonistic effects of these two solutions on histamine [H.sub.1] receptors. The results of this group also indicate anticholinergic and/or adrenergic stimulatory effects for macerated extract and essential oil. The non-significant difference between the values of (CR-1) obtained in the presence of both solutions in this part of the study with that of chlorpheniramine indicates comparable antagonistic effect of the solutions relative to chlorpheniramine at concentrations used. Although improvement in maximum responses was obtained in the presence of macerated extract and essential oil in group 2 experiments compared to those of group 1, there were still significant differences between maximum responses obtained in the presence of both solutions and that of saline, indicating small functional antagonistic effects of these solutions at histamine [H.sub.1] receptors other than [beta]-adrenergic stimulatory and/or muscarinic blocking effect.

In order to investigate whether changes of concentration-response curves observed in group 2 are due to [beta]-adrenergic stimulatory or muscarinic blocking effects, the antihistaminic effect of the plant was also examined on tracheal chains incubated with indomethacin and propranolol. The results of this part of the study obtained in the presence of macerated extract were fairly similar to those of group 1 and those obtained in the presence of essential oil were similar to those of group 2 study. These results indicate that functional antagonism of macerated extract at histamine [H.sub.1] receptors is mainly due to the blocking effect of this extract on muscarinic receptors. Non-parallel shift in histamine-response curves obtained in the presence of macerated extract in groups 1 and 3 could be due to the presence of substances with muscarinic receptor blocking effect and also substances with competitive antagonistic effect at histamine [H.sub.1] receptors in this extract from B. persicum. The results obtained in the presence of essential oil and aqueous extract in this part of the study showed that the functional antagonism of these two solutions observed in group 1 experiments is mainly due to stimulatory effect on [beta]-adrenergic receptors. In a previous study, we have also demonstrated a competitive antagonistic effect of macerated extract and a noncompetitive antagonistic effect of aqueous extract and essential oil from B. persicum at muscarinic receptors (Boskabady and Talebi, 1999) which confirm the results of the present study. The values of (CR-1) obtained in the presence of both solutions in this part of the study were not significantly different from that of chlorpheniramine indicating comparable antagonistic effect relative to chlorpheniramine at concentrations used.

In order to inhibit arachidonic acid metabolism, in all three parts of the study, tissues were incubated with indomethacin. The different histamine [H.sub.1] receptors blocking effect of extracts is presumably due to the variation of methods of extraction. The lower [H.sub.1] receptors blocking effect of essential oil compared to the effect of extracts is presumably due to its solubility in water of organ bath.

In conclusion, the results of this study suggested a competitive antagonistic effect of B. persicum at histamine [H.sub.1] receptors. In addition, the results indicated a blocking effect of macerated extract from this plant at muscarinic receptors, a stimulatory effect of essential oil and aqueous extract on [beta]-adrenergic receptors.
Table 1. E[C.sub.50] ([micro]M) of histamine in the presence of aqueous
extract, macerated extract, essential oil, 20 nM chlorpheniramine, and
saline in three groups of experiments

Solutions Group 1 Group 2

Saline 17.00[+ or -]3.88 22.80[+ or -]2.72
 ns
Aqueous extract 37.25[+ or -]4.94 49.88[+ or -]3.63
 * **** ns
Macerated extract 45.13[+ or -]5.80 50.50[+ or -]2.60
 *** **** ns
Essential oil 35.75[+ or -]5.36 45.50[+ or -]2.35
 * **** ns
Chlorpheniramine 39.13[+ or -]3.92 45.00[+ or -]3.93
 *** **** ns

Solutions Group 3

Saline 27.75[+ or -]2.48
 ns nS
Aqueous extract 45.63[+ or -]3.85
 **** ns nS
Macerated extract 60.88[+ or -]2.79
 **** [iota] nS
Essential oil 53.63[+ or -]4.84
 **** [iota] nS
Chlorpheniramine 53.63[+ or -]4.61
 **** ns nS

Values are presented as mean [+ or -] SEM. Group 1: experiments on
tracheal chains incubated with 1.4 [micro]M indomethacin; group 2:
experiments on tracheal chains incubated with 1.4 [micro]M indomethacin,
1 [micro]M propranolol and 10 nM atropine; group 3: experiments on
tracheal chains incubated with 1.4 [micro]M indomethacin and 1 [micro]M
propranolol (for each group, n = 8).
Significance of differences: (1) plant solutions vs. saline: NS:
nonsignificant difference; *: p<0.05; **: p<0.01; ***: p<0.005; ****:
p<0.001, (2) the results of groups 2 and 3 vs. group 1; ns:
nonsignificant difference; [iota]: p<0.05; [iota][iota]: p<0.001, (3)
the results of group 3 vs. group 2; nS: non-significant difference;
[epsilon]: p<0.05; [epsilon][epsilon]: p<0.001.

Table 2. Slope of histamine log concentration-response curves in the
presence of aqueous extract, macerated extract, essential oil, 20nM
chlorpheniramine, and saline in three groups of experiments

Solutions Group 1 Group 2

Saline 1.3[+ or -]0.13 1.57[+ or -]0.09
 ns
Aqueous extract 0.76[+ or -]0.06 1.00[+ or -]0.14
 *** * ns
Macerated extract 0.62[+ or -]0.08 1.34[+ or -]0.11
 *** NS [iota][iota]
Essential oil 0.80[+ or -]0.16 1.37[+ or -]0.09
 * NS [iota]
Chlorpheniramine 1.26[+ or -]0.12 1.56[+ or -]0.15
 NS NS ns

Solutions Group 3

Saline 1.57[+ or -]0.08
 ns nS
Aqueous extract 1.40[+ or -]0.09
 NS [iota][iota] nS
Macerated extract 0.81[+ or -]0.10
 **** ns [epsilon][epsilon]
Essential oil 1.40[+ or -]0.15
 NS [iota] nS
Chlorpheniramine 1.46[+ or -]0.07
 NS ns nS

For abbreviations see Table 1.

Table 3. Maximum response to histamine obtained in the presence of
aqueous extract, macerated extract, essential oil, 20 nM
chlorpheniramine, and saline in three groups of experiments

Solutions Group 1 Group 2

Saline 100.0[+ or -]0.0 100.0[+ or -]0.0
 ns
Aqueous extract 53.75[+ or -]5.36 59.00[+ or -]6.19
 **** **** ns
Macerated extract 43.06[+ or -]6.20 72.38[+ or -]7.16
 **** ** [iota][iota]
Essential oil 59.06[+ or -]10.5 64.75[+ or -]5.40
 ** **** ns
Chlorpheniramine 91.50[+ or -]3.02 87.75[+ or -]3.60
 NS NS ns

Solutions Group 3

Saline 100.0[+ or -]0.0
 ns nS
Aqueous extract 74.38[+ or -]3.87
 *** [iota] nS
Macerated extract 50.00[+ or -]3.74
 **** ns [epsilon]
Essential oil 74.88[+ or -]5.36
 *** ns nS
Chlorpheniramine 91.75[+ or -]2.63
 * ns nS

For abbreviations see Table 1.


Acknowledgements

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

Received 3 February 2003; accepted 26 June 2003

References

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Arunlakshana, O., Schild, H.O., 1959. Some quantitative use of drug antagonists. Br. J. Pharmacol. 14, 48-58.

Avesina, A., 1991. Law in Medicine, Vol. 2, 2nd Edition, A. Sharafkandi, translator. Soroush Press, Teheran, pp. 141-142.

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Boskabady, M.H., Talebi, M., 1999. Bronchodilatory and anticholinergic effects of Carum carvi on isolated guinea-pig tracheal chains. Med. J. I. R. Iran 12, 345-351.

Holroyde, M.C., 1986. The influence of epithelium on the responsiveness of guinea-pig isolated trachea. Br. J. Pharmacol. 87, 501-507.

Linden, A., Bergendal, A., Ullman, A., Skoogh, B.E., Lofdahl, C.G., 1993. Salmetrol, formetrol, and salbutamol in the isolated guinea-pig trachea: differences in maximum relaxant effect and potency but not in functional antagonism. Thorax 48, 547-553.

Popa, V.T., 1977. Bronchodilatory activity of an [H.sub.1] blocker chlorpheniramine. J. Allergy Clin. Immunol. 59, 54-63.

Sardari, S., Armin, G.R., Micetich, R.G., Daneshtalab, M., 1998. Phytopharmaceuticals, Part 1. Antifungal activity of selected Iranian and Canadian plants. Pharm. Biol. 36, 180-188.

Syed, M., Hanif, M., 1985. Antimicrobial activity of the essential oil of the Umblliferate family. Part 1. Cuminum cyminum, Coriandrum sativum, Foeniculum volgare and Bunium persicum oils. Pakis. J. Sci. Ind. Res. 55, 116-120.

M.H. Boskabady*, A. Moghadas

Department of Physiology, Ghaem Medical Centre, Mashhad University of Medical Sciences, Mashhad 91735, Iran

*Corresponding author. Tel./fax: +98-511-841-3579.

E-mail address: mhboskabady@hotmail.com (M.H. Boskabady).
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Title Annotation:research findings
Author:Boskabady, M.H.; Moghadas, A.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Jul 1, 2004
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